Lecture 13. Protein Function and Enzymes.

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Lecture 13. Protein Function and Enzymes.
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Lec 13. Introduction to Chemical Biology -- Protein Function and Enzymes
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UCI Chem 128 Introduction to Chemical Biology (Winter 2013) Instructor: Gregory Weiss, Ph.D. Description: Introduction to the basic principles of chemical biology: structures and reactivity; chemical mechanisms of enzyme catalysis; chemistry of signaling, biosynthesis, and metabolic pathways. Index of Topics: 0:00:41 Enzymes 0:03:20 Repeat Proteins 0:05:16 Equilibrium Constants 0:06:25 Enzymatic Catalysts = Catalytic Receptors 0:07:03 Michaelis Constant for Measuring Catalysis 0:15:30 The Perfect Enzyme 0:21:29 Kinases: Phosphorylation of Ser/Thr or Tyr 0:34:19 Why Study Single Molecules? 0:37:44 How to Follow Enzymatic Catalysis with Single Walled Carbon Nanotubes 0:41:34 Single Biomolecule Bioelectronics 0:44:24 Before and After Enzyme Attachment 0:46:29 Watching cAMP - Dependent Protein Kinase A 0:49:18 Further Generalization: Protein A Kinase 0:55:55 Lysozyme as a Model Enzyme for Glycosdie Hydrolysis 1:08:22 Proteases Cleave Amide Bonds 1:12:37 Regulation of Proteases Through Pro-Enzymes
Übergangszustand Activation energy Reaction mechanism Activity (UML) Binding energy Ligand Nuclear receptor Collagen Man page Titanium Meat analogue Enzyme Invar Cell (biology) Protein Essenz <Lebensmittel> Insulin Genregulation Fruchtsaft Butcher Lambic Motion (physics) Transition metal Setzen <Verfahrenstechnik> Mashing Assembly (demo party) Chemical reaction Mutagenese Electronic cigarette Meat analogue Protein Functional group Enzyme Optische Analyse Genregulation Cell (biology) Chemical structure Dissoziationskonstante Repeated sequence (DNA) Motion (physics)
Sense District Area Collagen Octane rating Topicity Protein domain Wursthülle Chemische Biologie Protein Hydroxyprolin Protein Umweltchemikalie Turbulence Chemical structure Repeated sequence (DNA) Helix Sea level Helix Repeated sequence (DNA)
Peptide Eau de Cologne Elektrolytische Dissoziation Proline Protein domain Nuclear receptor Wursthülle Wine tasting descriptors Enzyme Protein Active site Antigen Stuffing Process (computing) Controller (control theory)
Atom probe Antibody Octane rating Finings Binding energy Operon Wursthülle Nuclear receptor Solution Man page Zeitverschiebung Enzyme Volumetric flow rate Tidal race Cupcake Shear strength Cell (biology) Mortality rate Ausgangsgestein U.S. Securities and Exchange Commission Transition metal Setzen <Verfahrenstechnik> Substrat <Chemie> Elektrolytische Dissoziation Octane rating Gleichgewichtskonstante Multiprotein complex Chemical reaction Chemistry Phosphatase Gesundheitsstörung Reaction rate constant Enzyme Peroxidase Substrat <Chemie> Controller (control theory)
Glucose Decomposition Digoxigenin Bohrium Erdölraffination Proteindesign Electrical breakdown Metabolism Binding energy Molecule Enzyme Cupcake Concentrate Octane rating Mashing Chemical property Chemist Katalase Multiprotein complex Chemical reaction Erdrutsch Wine tasting descriptors Reaction rate constant Dissoziationskonstante Cobaltoxide Wasserbeständigkeit Stereoselectivity Antigen Sense District Ionenbindung Reaction mechanism Watt Cytochrome Hydrogen peroxide Wursthülle Man page Zeitverschiebung Cell (biology) Active site Ausgangsgestein Oxide Gravel Glasses Peroxide Macintosh Spawn (biology) Biochemistry Water Gesundheitsstörung Initiation (chemistry) Functional group Pinot noir Substrat <Chemie> Soap
Setzen <Verfahrenstechnik> Cell fusion Synthase Radioactive decay Enzyme Tidal race Protein Lyasen Posttranslational modification Toxin Oxide Periodate Decarboxylasen Octane rating Katalase River source Multiprotein complex Chemical reaction Erdrutsch Blue cheese Enzyme Azo coupling Proteom Dielectric spectroscopy Thermoforming Stuffing Sense District Ionenbindung Biosynthesis Sprayer Aldosterone Cytochrome Wursthülle Ultraschallschweißen DNS-Synthese Man page Magnetometer Cell (biology) Controller (control theory) Common land Mortality rate Smoking (cooking) Amino acid Active site Elektronentransfer Sea level Glycosylation Substrat <Chemie> Arginine DNS-Synthese Gleichgewichtskonstante Ubiquitin Cigarette Opal (programming language) Korken Water Hydrogen Ice Epoxidharz Gesundheitsstörung Functional group Chemical structure Substrat <Chemie> ISO-Komplex-Heilweise
Activin and inhibin Protein domain Phosphorous acid Binding energy Sulfur Alpha particle Avidity Molecule Adenosine triphosphate Enzyme Protein Phosphate Insulin Posttranslational modification Enzyme inhibitor Chemical reaction Erdrutsch Hydroxide Wine tasting descriptors Thorium Tyrosin Phosphate Enzyme Aage Cobaltoxide Ionenbindung Bearing (mechanical) Activity (UML) Reaction mechanism Hydroxyl CHARGE syndrome Wursthülle Nuclear receptor Soil conservation Lewisite Man page Magnesium Meat analogue Hydroxybuttersäure <gamma-> Active site Beta sheet Elektronentransfer Cobaltoxide Stickstoffatom Substrat <Chemie> Malerfarbe Man page CHARGE syndrome Meat analogue Magnesium Functional group Elektronentransfer Salt Chemical structure Substrat <Chemie>
Hydroxyl CHARGE syndrome Chemical reaction Lewisite Adenosine Erdrutsch Magnesium Acid Magnesium Phosphate Hydroxybuttersäure <gamma-> Phosphate Elektronentransfer Elektronentransfer Chemical structure Carboxylierung
Residue (chemistry) Activity (UML) Erdölraffination Binding energy Set (abstract data type) Wursthülle Cyclo-AMP Block (periodic table) Anomalie <Medizin> Molecule Cell division Enzyme Protein subunit Cell (biology) Protein Tandem-Reaktion Explosion Elektronentransfer Active site Sea level Process (computing) Regulatorgen Concentrate Stockfish Death Meat analogue Phosphate Metabolic pathway Blue cheese Azo coupling Genregulation Stuffing
Sense District Residue (chemistry) Activity (UML) Finings Wursthülle Molecule Meat analogue Enzyme Hydroxybuttersäure <gamma-> Active site Sea level Motion (physics) Area Metal Stickstoffatom Regulatorgen Molekulardynamik Organic semiconductor Chemical reaction Multiprotein complex Wine tasting descriptors Meat analogue Biochemistry Combine harvester Chemische Biologie Acid Tyrosin Genregulation Breed standard Inhibitor Base (chemistry) Stuffing
Ionenbindung Cell growth Enzymkinetik Copper Wursthülle Nuclear receptor Man page Molecule Carbon nanotube Meat analogue Enzyme Magnetometer Biophysics Tool steel Storage tank Motion (physics) Area Surface science Chemical property Spring (hydrology) Fluorescence Walking Molekulardynamik Carbon (fiber) Gold Electronic cigarette Wine tasting descriptors Molybdenum Water Chemische Biologie Functional group Transdermales therapeutisches System Acepromazine Proton-pump inhibitor Dye penetrant inspection Chemiefaserverstärkter Kunststoff
Sense District Flame Coiled coil Magnetism Functional group Protein Lightning CHARGE syndrome Wine tasting descriptors
Gluten Attachment theory Enzyme Enzyme Protein Carbon (fiber) Gold Addition reaction Wine tasting descriptors Man page
Grading (tumors) Radiation damage Sunscreen Growth medium Katalase Gold Wursthülle Water Molecule Attachment theory Enzyme Gesundheitsstörung Electron Enzyme Azo coupling Cell (biology) Protein Common land Salt Polymer Library (computing) Mixing (process engineering) Stuffing
Glucose Digital elevation model Bohrium Erdölraffination Metabolism Memory-Effekt Anomalie <Medizin> Molecule Carbon nanotube Enzyme Phosphate Surface science Mashing Walking Sulfur Katalase Food additive Hydroxide Systemic therapy Vancomycin Attachment theory Phosphate Protein Enzyme Optische Analyse Hydroxybuttersäure <gamma-> Cell (biology) Dissoziationskonstante Zunderbeständigkeit Cobaltoxide Pipette Stuffing Sense District Sodium hydride Activity (UML) Sunscreen Peptide Enzymkinetik Wursthülle Man page Meat analogue Fermentation starter Hydroxybuttersäure <gamma-> Cell (biology) Essenz <Lebensmittel> Active site Sea level Molecularity Substrat <Chemie> Spawn (biology) Buffer solution Water Song of Songs Sea level Functional group Genregulation Elektronentransfer Substrat <Chemie>
Kohlenhydratstoffwechsel Bohrium Polysaccharide Hydrolase Molecule Carbon nanotube Glutamic acid Tiermodell Enzyme Electron Library (computing) X-ray crystallography Surface science Headache Concentrate Gas Chemist Chemical reaction Acid Enzyme Hydrolase Cell (biology) Locus (genetics) Alu element Cobaltoxide Sense District Ionenbindung Blood vessel Reaction mechanism Enzymkinetik Wursthülle Man page Neutralization (chemistry) Cell wall Lysozyme Cell (biology) Common land Active site Motion (physics) Conformational isomerism Polysaccharide Molekulardynamik Ethylene-vinyl acetate Glykoside Biochemistry Water Functional group Smoky quartz Substrat <Chemie> Base (chemistry) Carboxylierung Till ISO-Komplex-Heilweise
Conformational isomerism Insulin shock therapy Setzen <Verfahrenstechnik> Coral reef Density Polysaccharide Lysine Erdrutsch Wine tasting descriptors Rapid Enzyme Volumetric flow rate Magnetometer Enzyme Lysozyme Controller (control theory) Cell (biology) Suspension (chemistry) Substrat <Chemie> Periodate Motion (physics)
Residue (chemistry) Sodium hydride Activity (UML) Polysaccharide Enzymkinetik Man page Wine tasting descriptors Enzyme Chemische Synthese Lysozyme Controller (control theory) Active site Substrat <Chemie> Peptide Proline Animal trapping Anomalie <Medizin> Plant breeding Genotype Rapid Enzyme Attitude (psychology) Substrat <Chemie> Carboxylierung Thermoforming Stuffing
Sense District Ionenbindung Blood vessel Nitrogen fixation Reaction mechanism Peptide Sunscreen Radiation damage Polysaccharide Wursthülle Cross-link Man page Origin of replication Enzyme Lake Rapid Protease Lysozyme Cell (biology) Tandem-Reaktion Genregulation Butcher Surface science Whitewater Substrat <Chemie> Peptide DNS-Synthese Plant breeding Protein Blue cheese Dielectric spectroscopy Substrat <Chemie>
Surface science Reaction mechanism Cytochrome Papain Gene Genotype Wine tasting descriptors Man page Carbon nanotube Chromosome Protease Enzyme Cell (biology) Clay Cell (biology) Cysteine
Sense District Übergangszustand Residue (chemistry) Ionenbindung Reaction mechanism Bohrium Man page Hydrogen Molecule Enzyme Electron Protease Active site Cysteine Ionenbindung Octane rating Protonation Multiprotein complex Electronic cigarette Acid Functional group Enzyme Proton-pump inhibitor Elektronentransfer Cobaltoxide
Proline Recreational drug use Serine Ligand Acid Lewisite Man page Molecule Enzyme Protease Active site Genregulation Cysteine Process (computing) Area Set (abstract data type) Peptide Precursor (chemistry) Physiology Seafloor spreading Chemical reaction Electronic cigarette Functional group Enzyme Elektronentransfer Transformation <Genetik>
Transition metal Übergangszustand Nahtoderfahrung Setzen <Verfahrenstechnik> Enzyme inhibitor Enzyme inhibitor Bohrium Phosphorous acid Patent Wine tasting descriptors Man page Meat analogue Block (periodic table) Meat analogue Enzyme Protease Protein Enzyme Hydrolysat Freies Elektron Zunderbeständigkeit Inhibitor Common land
we're going to pick up where we left off of you hopefully you watch the podcasts lecture on Tuesday and I want to pick up what we discussed on Tuesday protein function as specifically wouldn't be talking about how enzymes work had 2 enzymes catalyze the reactions in the cell and so on were going to
start the show by talking about some measures of enzyme activity and then we'll talk about regulation amends lines and the will get into the mechanisms and will close with mutagenesis an engineering OK so what we talked about it on Tuesday Is proteins and have a wide range of roles structural binding and catalytic roles in example a structural we saw for example collagen and we saw how college gets organized into these complex assemblies that make it possible to have extremely strong bones and things like that and we have to talk a little bit about Titan muscle protein that makes muscles capable of being pulled through stretched from without breaking without snapping out we also talked about finding the example we saw there was at the ATP binding to FDA 506 and rapper license so and then finally we're now at the part start talking about catalysis so protein function has at least 3 major roles inside the cell structure finding and catalysis and when we talked about the repeat proteins for example and we saw a great example of binding and I wanna just very briefly will will take a look at that at the moment but just 1 emphasize something based on some questions that I got there in my office hours and we talked about how non-covalent receptor like interactions can be described by dissociation constants and an honorary and off we're now at the point where Oregon start talking about this Michaelis-Menten constant payout which is an analog to the and something that I touched on the very end of the lecture is enzymes work by lowering the transition state enzyme of the reaction energy of the reaction during this is in essence how you catalyze reaction lowering the activation energy necessary for that reaction to take place you're increasing the speed that the reaction takes place by doing this and you do then enzymes do this by binding to the transition state and stabilizing by having our counter alliance that stabilize charred functionalities in the transition state the enzyme can lower the transition state energy and this is surprisingly effective we're going to be seeing some examples that so we're going to be talking today also about health this catalysis by enzymes is coupled to the motion by the enzyme and I will show off trees some examples of best and what we're going to see Is that enzymes force the substrate starting material into confirmations that favor formation of the product and by doing that that accelerates these reactions OK and very briefly just wanna touch on 1
point about the repeat
proteins something I didn't mention that probably should have won I talked about the peak ratings dissident example of repeat protein an anchoring repeat notice that it has a series of Felix turning Felix turning Felix territory Felix turned like Terry Ulick turbulence so those repeats each 1 of these New movements and then he likes me and then he likes to notice that all stacked on top of each other each 1 of those is 1 example of a repeat and this anchoring repeat as a whole bunch of these lined up and that's the work gets the name repeat protein makes sense and then similarly of pollution which repeats in this case it's a helix stranded Felix stranded live feel extremely etc. a series of repeating motif repeating structural and in fact these are actually individual domains the full modestly well on their own and they can be shuffled around to a limited extent it's OK any questions about this that the topic of protein structure and for that matter any questions about anything was covered on Tuesday in Tuesday's lecture areas like I think in that case were ready to move on and we're going to talk next about catalysis today's discussion is going to be pretty high level I'm going to be telling you stuff that's actually not in the book and in fact actually it's only found really the frontiers of of of literature and chemical biology so don't hesitate to interrupt if at any point I start to lose you OK it's better if you interrupt me early on that if I go further down the road in your totally lost a because the truth is this electoral dutifully time you're going to be all find this material be discussing it and I think it's very very important it's actually the ferry frontiers of chemical biology case I want to talk to
you today about catalysis and
last time I showed you that non-covalent binding consists of like against hopping on to some binding site in a receptor and we describe this non-covalent interactions using an equilibrium constant especially equilibrium constant called the dissociation constants that I had that quantifies the Riccio between unbalanced appear to be bound by receptor alike and interactions case this is pretty straightforward stuff the only difference with catalysis is ever going to have a similar receptor like into direction but upon binding to light the and the enzyme in this case instead of the receptor and for lacking instead of the receptor the lighting and is going to be transformed to converted into some new products so it's a similar sort of process we understand non-covalent interactions that we can also understand catalysis by enzymes OK I'm
skipping ahead skipping
skipping we talked about this already parent this is I
believe this is where we left off this straight OK so where we left off again is this idea that I receptor like interactive attractions are governed by some binding in the case of enzymes the substrate fines and bans on some catalysis takes place to convert the as substrate into a product S stands for substrate for starting material and that gives us P which is the product and then the product tested associate I think I've talked about
this race this was covered by a cover this up again right OK
so I think I'm actually over here OK so here's a typical reaction seemed diary talked about on the previous so the formation of some of this of this enzyme substrate product this Micaela cemented complex is sometimes called a Michaelis-Menten complex this was this complex here of the activated inside down to the substrate in a way to catalyze the reaction of has equilibrium constants and in the same way that the KGB was proportional to the rate constants for the dissociation that chaos to carry on in the same way the Michaelis-Menten equilibrium constant is equal to these are some of the operator and the cake hat so that's like getting to the and going backwards or forwards that's the rates going New Directions for destruction of the complex Everson warming the complex being the reconstituted for K on this payout resembles the Katie Ford non-covalent into binding interactions and so it's useful for us the strength of the case tells us something about how avidly the enzyme is going to grab onto the substrate and how quickly exploited for this fight yes complex so that that this stadium is actually a useful number it tells us something about the conditions inside the cell because the enzyme has to evolve up to a
sufficient affinity for its substrate to glass onto the substrate inside the cell if the affinity is too low then on the enzyme substrate complex will never form an enzyme liver catalyze reactions on the other hand if it evolves to the point where it's uh you know super-duper high maybe that's not so useful for the Celtics native enzyme there will be a little too active so he's a ball for up to the maximum the ability that's necessary for the cell required for the conditions found inside the cell and often times for example we can engineer enzymes have very different camps simply by tinkering with their active sites and I'll talk about that more in a moment when we talk about protein engineering OK so if we look at the AI if we look at a dose-response diagram this is similar to the dose-response diagrams they showed on a previous slide on Tuesday instead on the Y axis we have our initial reaction balls lost city and the concentration of substrate the point of inflection here is going to be roughly the payout this equilibrium constant up here and for that matter the and there are at this point of inflection also tells us that we're 50 per cent of the maximum rate of the enzyme is going to be at the very highest concentrations of substrate the enzyme is going to be running flat out OK so that's like as fast as the enzyme can possibly go on be the equivalent of giving a sprinter maximum oxygen at maximum because everything here she needs to do to run as as fast as possible so that's up here under maximum velocity conditions notice that this asymptotic Lee approaches this the max value way up here and so did anything works at excess concentration of substrate that's called the Mac's conditions and typically enzyme reactions that run in the laboratory are run under those conditions we always have a extreme excesses substrate typically it's OK let's
take a look at some parents say they widen the range widely there's a wide range of possible payouts for enzymes and I hear some numbers over here now like the KDE a lowercase and value means tighter binding totally analogous to the dissociation constant in fact it's it's has a very similar connotation so what this tells us for example is that if this enzyme here cytochrome P 4 50 binds spend soap I read with very high affinity this should come as no surprise to us right bands of Pyrenees as Big Flat hydrophobic molecules and hydrophobic molecules in general so soluble so the cytochrome P 4 50 in your liver is going to be grabbing on to the Denzel pirates at you inhaled on your way to over here when he got behind that stupid shuttle bus that was you know digging along the slower speed write you get behind the exhaust pipe that shuttle bus and he started feeling on bird bands of pirates cytochrome P 4 50 delivers right now as we speaking ratting on with great event that for for the bands of pirates on the other hand there some enzymes that don't have the gravel on all that well to the substrates like a connotation this is a key enzyme in the metabolism of glucose and I it's that's it's substrate citrate is found in high enough concentrations that enzyme doesn't really have to evolve to a very high affinity so this gives us sort of a crude measurement of hat with the concentration of the substrate is in cell right so what we know is that is probably not a lot of them so tiring president but there's probably tons of citrate present hence the need for lower affinity now let's also take a look at some hats so this is the equal of this is the rate constant for the decomposition of the Michaelis-Menten complex the E S complex that is now being broken down to perform enzyme product it makes sense OK so In this case again there's a wide range of cats and this tells us something about how hard the reaction is the catalyze harder reactions in general have slipped lower K cats but I can also tell us something about and evolution of enzyme enzymes in general evolve up to the required functions and really don't go past that apparently there's really no there's no evolutionary dried fish no selection mechanism that drives the enzymes to be perfect unless they need to be perfect for some particularly crucial function for the cell so here is 1 example really crucial functions for the cell the enzyme catalyzed breaks down hydrogen peroxide into oxygen and water this is a crucial are reaction hide your peroxide creatures that there's a substantial burden on cells this is isn't a strong oxidant and oxidants run around and wrecked havoc on cellular machinery and so for this reason sales have evolved pretty sophisticated mechanisms to very quickly break down such oxidation products and I can always has a cake cat of a 100 million so this is a really really fast the catalytic reaction that it had that takes place on and then a slower reaction would be appropriated producers of course high July stammered bonds I believe we've seen these before and I had their pay cats are much lower likely because this reaction is a little more challenging and a a little less favorable but thermodynamic play and also for that matter it's not as critical perhaps for the cell OK question so far good OK said these are the numbers that are going to underlie a discussion as we start talking about the properties of enzymes and this is this the same numbers that you learn about it like bio 99 a 98 whatever biochemistry class your Vidor elsewhere these these numbers are kind of the vocabulary that the by biochemistry friends talk about when they talk about enzymes cake now that the truth is as a chemical biologists don't get too worked up about these numbers are more interested in understanding the Adams and bonds basis for held enzymes work and so I
guess the best place to start would be let's start with the perfect what would be the enzyme that really can crank that could maximize its of its ability to turn overreaction and they will look into it some specifics at the level about bonds so the very perfect enzyme you might imagine every time it forms this Michaelis-Menten complex the ES complex then it goes immediately take a cap so it forms and in blue it's over to a cat and is immediately gets converted to converted converts the substrate to the product that happens instantly on the other hand the perfect enzyme is not going to have any offering over here this offering represents lost opportunities this is the see the wasteland of couldn't could additional of maybe should have a right this is the chance that the enzyme next so instead of going to products enzyme goes backwards and so this operate over here is a miserable in inefficient for an enzyme so the perfect enzyme is not going to have an offering and so the perfect inside you can basically imagine playoff being 0 and if we have that and then we can imagine of rearranging Arcadian equation just a couple of slides ago such that K R equals the ratio of the capped to carry and again notice these little cases are indicating rate and the decay emissary equilibrium constant so the very best enzyme will have an armed raid that's diffusion controls in other words is limited by the amount of time that the substrate Brownian motion style eventually bounces his way to the active site that's should be the slowest at 4 it enzyme that's perfect and we talked about this before but that rated the fusion has a speed limit of 10 tonight for Moeller per 2nd can't go any faster than that of physical or it's like the speed of light you cannot exceed that of the Jim just because it takes a little while to bounce around through all that water and other stuff that's present in the cell it makes sense and will take a look at a moment at an example of an enzyme that's far from perfect and will start to understand what its sources of imperfection are so
do me just give you a little take table that I really like that shows us and helped us organize enzymes that shows us the rankings of enzymes in your proteome they said this is a listing from most common to these comments like a greatest hits of the 7 categories of enzymes that are found in the human podium the most common and signed by far are the high delays as the ease of enzymes that introduce water as a way of breaking up on and we're going to see a couple of examples of this will see examples of glycosylation is and producers today we've already seen examples of nuclear uses that was stuff like artists right number we we talked about are and was inhibited by Dempsey this is a similar sort of thing can someone help this guy off thinking OK transfer races next most common seconds seconds position and these are examples of enzymes that transfer functionality from 1 spot to another and we're going to look in detail at an example of a protein kind today and then later in the class will look at it like Castleton sprays oxide adopted says this is like the enzyme cytochrome P 4 50 that takes the bands of Pyrenees introduces an epoxy and oxidises the bands of pirates you all remember this right I showed you the men's apparel couple slides ago but earlier this quarter Boehner's talking about cigarette smoking I showed you how the band's umpiring that looks like this rather innocuous black structure gets converted into an epoxy and then slips into the pie stack of UGA calculates that unit and so these are actually very common and signs the outsider reductase is because they're important for removing toxins on the hydrogenation is another 1 that's very common and perhaps will get a chance to see this 1 today on and then finally we get down to the light cases these are enzymes that the spot weld together to functional groups such as attaching ubiquitin DNA to something by some races are used to convert substrate into some related ice America products but these are things like appearances on the said the as we've seen before we talked about always ulterior said the taste this was that gargantuan on complex that ran out the unity code on and the various modifications of the T to make sure that the correct amino acid was being attached to the RDA during an eagerly to urinate synthesis peso and then the final 1 the wildlife users are doing things like the carboxyl Asian actually breaking carbon-carbon bonds in dramatic fashion to these are all the lasers they're doing Alvalle reaction etc. period breaking or making carbon-carbon bonds so fairly like we've seen many examples of these different enzymes this quarter so now I can go through and just talk about the ones that are really important that we haven't seen yet a case such as the kind cases over here and I believe that's what I'm going to start yes so it turns out that kind
cases have a common cold that consists of a lower domain down here and then a lodges of but appears to the active site is indicated where these manuals on this is TP so please is take ATP and transfer the gamma phosphate of ATP to some sort of hydroxide recipient and that's generically what they're doing but when we talk about the game of prostate ATP adenosine triphosphate had the ATP has 3 parts groups called alpha beta and gamma the 3rd 1 in the row is called gamma and so that's phosphate groups that kind cases are going to transfer so again notice that these have a conserved Abdul Loeb structure even know these have widely disparate activities this is everything from a receptor tyrosine kind over here but to protein coyotes over here these stew of these do have very different activities that across early different targets and out yet another here and they all of all have very similar structures now
this class of enzymes like all enzymes can be inhibited by substrates pseudo substrates that mimic the real substrate so here's the structure of ATP even better place of 1 of the oxygen of ATP highlighted in blue we have nitrogen and this phosphor avid inhibits the kind that said you feed this this past gravity to tiniest any of the kind I showed him the previous slide it's going to be game over for them they're not going to be able to work because they're going to this this ATP analog the can and then put it sloppy old bear hug but this cannot phosphate is missing the oxygen and missing oxygen is the same as saying yes totally in and so this is going to basically be locked in active site in the brief said that this site yet unable to transfer the phosphate groups and so the net effect is to shut down and sign and this is a very effective way of killing enzymes you basically know something about the mechanism you make a tiny little modification of substrate and boom it's game over for a day enzyme OK so that you could do this also was sold for or nitrogen and so I have shown you attributed to those well and again it's 6 inapposite inhibits enzyme in this at this approach also works if you mimic the product and a large number of enzyme inhibitors pursue 1 of those 2 approaches mimicking the substrate as shown here are mimicking the product you approach works great so that Suleman I showed you the structure of the by structure of Chinese that and take a look at its active site in the active site there are around here's the structure of ATP there are a series of conserved magnesium ions these balls over here that are bound to the phosphate groups of the ATP the numbers here indicated Angstrom the distances paintings numbers are pretty low right if you recall the carbon-carbon bond is somewhere on the order of like 1 . 5 extra inches so these are pretty close in numbers right these this magnesium is getting awfully close to this oxygen over here and these are cozy cozy molecules and that's the likely disclosed they like being this close because they have a complementary charges right magnesium has at last to charge the oxygen the spot had negative charges so there are attracted by salt bridges or cool loans interactions that we saw earlier in the quarter so over here there is the these other substrate for this enzyme reaction that has a hydroxyl showing it to you with the hydroxyl deep-rooted aided and after Apostol transfer this oxygen the substrate has now picked up a pass the groups and noticed that the man these things here are helping to stabilize that phosphate right that they're lowering energy of binding by forming that same sort of colonic salt bridge that we saw earlier OK now the zoom in and take a look at the pushing the bearer pushing mechanisms for this enzyme active-site what we find is that and not depicted on
this previous slide somewhere out here there's a
carboxyl late from Spartacus at the Curragh box later this despotic acid deeper relates the Proton of the hydroxy of the scene of a series for the substrate and that sets us up with that Alcock side up Cox idea superb nucleophile it's negatively charged can attack the gamma prostate of ATP and again the magazines get in on the action there over here participating fully and stabilizing this negative charge of phosphate group that's crucial break you can imagine this reaction not going in the absence of those magnesium it's great because 1 negative charge is not going to want to approach a negatively charged past secret right the negatively charged Alcock side over here is going to be stymied is in its attack it's going to get repelled by this fast career so the main museums are shielding the phosphate groups and protect again and preventing it from getting from looking like a negative charge and said that tees up this reaction very deeply and then finally there's a collapse of this traditional biker middle intermediate and I just very briefly the structure around the spot say looks like a trickle by pyramid we have not so anyway that's interesting and there's collapses triggered a wider Middle intermediate to give us the final product OK so to summarize the most important aspect of this is the notion that the magnesium ions are playing several roles to make this reaction possible 1st there and coordinating and stabilizing the transfer phosphate groups as a Lewis acid peso that helps accelerate the reaction as Lewis acid it turns out that
kind activity in the cell very tightly regulated and the reasons for this 1 or perhaps not clear if you don't know much about sets that signal transduction and understand very briefly covered today and then in the future lecture will learn quite a bit more about it In the cell there's a series of pathways that transfer information in these pathways are controlled by a transfer of tiny transfer of phosphate groups to key residues in proteins so kind cases player really key role in on kicking off various processes in the cell so there's cascades of kind cases where 1 kind it's possible relates the next kind which parts were later next pioneers and so on and so forth turns out that this process is very tightly regulated because you don't want yourselves going while you don't want them to be doing uncontrolled cell division for example and suffer this reason this cell very tightly regulates kind activities and I want to show you a couple of idiots about this but this tight regulation because it's crucial to understanding of how kind work OK so here is 1 example of this is an example from the enzyme protein kind as a as cyclic AMP regulated kindness and on the way this works is is actually a regulatory subunits shown here and below that lines to the kind and actually has been inhibitory move that blocks access to the active site case the other stock blue thing is binding here and has like this 1 finger that fits into the active site and blocks the kind of case from binding to any substrates the shuts down the kind it's an ability to show off the kind it is crucially important take if you don't have this kind is to be running around rampant wrecking havoc turning on stuff shutting Austin causing death and destruction and general mayhem and I do mean death and destruction of anti-Zionism are that important now when I'm the levels of a reporter molecule called cyclic AMP reaches certain concentrations this cyclic AMP binds to the regulatory subunit and causes the regulatory subunit to disassociate from the US the catalytic subunit approaching kindness and so these 2 molecules get forced apart as the blue 1 works into a new confirmation upon binding to cyclic AMP the thing changes its shape and it no longer has affinity for poaching kind he said this is that it is a variety Friday saying it reason not to go off and do the admission that its wanted to do for its entire life which is to run around the cell and fast for anything that moves nearly anything that moves it actually has a little bit of specificity but for the most part protein kind say what lots of likes the fossil relate what different binding partners because the pretty promiscuous molecules now
here is that there was another way of regulating enzymes is the fast relate them because this is
1 way we have some regulatory protein that binds a 2nd Way is
the boss for late residues better near the active site a case of for example this non-hydro Isobel analog of ATP which has a nitrogen in place locks to this is the molecule issued in a previous life on this child was active scientists but over here are to residues that could be possible related to football this on map kind basis P-38 gamma about kindness and so 1 of the these is a tyrosine another 1 is a series and so this kind is weeks around until it gets to the until it gets to a teller gets lost for elated at that point it goes into gear cases like an on off switch for the kind case in absence this then enzyme doesn't have the right confirmation doesn't have the right combination it can be a kind of cases of possible correlation of the kind is puts it in gear turns it on and sets a going makes sense any questions about what you've seen so far but that that's the basics Allen and talk to you about it really needs stuff that the latest results in thinking about how kind is his work and thinking about their own their emotions and again and this is kind of an abrupt departure from start standard material is presented in biochemistry classes and it really represents the frontier chemical biology and many of the next experiments talking about how we're done actually with with Mary she's 1 of the leaders of this area OK so the thing is I want to talk to you about Helen work at a mechanistic level and how they actually work dynamically has move when they do these reactions so I should tell you that enzymes have great emotions associated with their activities this is unlike the case of conventional catalysis by organic metallic complexes that you learned about backing 1051 a priority learned about income 125 in those cases there and get a metallic catalysts fines and Prasad place some Lewis acidic role but it's certainly we don't think about its motion and we don't think about it having some you know movement associated with its of dynamics in science it turns out for the most part almost all have very wild and very quick motions associated with them and a frontier chemical biology is to understand how those motions impact catalysis how did those motions of allowance signs to be effective catalysts and so on as and it turns out that if you get a big you know the round bottom flask full of enzymes you'll never be able to see those individual motions and the reason is they
tend to get word out of place so if we look at a large number of molecules will never see the individuals in motion because all of the enzymes in that flask bargaining running along at different speeds and everything gets Florida out OK so instead in order to see individual motions we have to look at single molecules and to understand this a little bit better money on offer an analogy let's imagine that I convinced them you know the Orange County marathon folks to rear out the marathon so instead of being on Sunday morning instead I convinced them to run here Thursday at 10 10 AM and I convinced them to start at that end of the classroom and said every all the runners through the classroom from that door to the store case and now everyone's running through here all 10 thousand runners you can imagine what you're going to see it's just a blur of pumping arms and legs right can be trying to get in and out of the classroom as quickly as possible as to be total pandemonium that's the situation when we look at an ensemble of enzymes it's total pandemonium it's a blur of arms and legs we don't see anything and everything gets averaged out and by CNG using tools like spectroscopy using tools and that's why you're familiar with from your other classes so over the last 15 years there's been a revolution in this area of chemical biology or biophysics where scientists has started to look at individual molecules in isolation from all of the other friends and neighbors Hasan now instead of having the entire marathon coming through the classroom let's imagine that I convinced each water to come running through 1 at a time so they're gonna start over there and they come running through here what you will see begins because each 1 is isolated from all the other runners is you'll you'll see their arms and legs moving right because now there's no blurring out effect right and furthermore if you look closely you'll be able to see some runners moving faster than others maybe 1 runner has a different stride then her neighbor right she comes running through and I I don't know maybe she she extends her legs a little longer than the runner behind her head but if we convinced them to be isolated from each other then we can really start to get information about how the move and that's a situation we find ourselves and when we start looking at enzymes and so this area single molecules allows us to look at conformational steps that intermediates and the kinetics and dynamics that underlined underlying enzymes function in this again is a major frontier is a really exciting area to be involved in research OK so everyone with me so far everyone understands the psyche of looking at single molecules right OK get and I want to talk to you next about how we're going to observe a single molecules there are a number of different fluorescence techniques for looking single molecules patch clapping you may have heard of it is a 40 year-old proven technology which really well for looking at individual receptors that works fine and in in the last 5 years or so groups here this year but have been at the forefront for inventing a sort of a tiny little microphone that allows us to listen 2 enzymes as they run and has some advantages over those other techniques and so that's what I want to talk to you about today OK so this
is a collaboration between my laboratory and Phil Collins and the Department of Physics you reduce your mind and he's pioneered ways of building circuits that are based on carbon nanotubes and I said this is an example of a carbon nanotube this is basically a carbon graphite and later that folded up into a cylinder head it looks sort of like chicken wire this these wires that are amusingly conductive carbon nanotubes are a really remarkable material they have remarkable mechanical properties they have a remarkable properties for conducting electricity and for conducting heat in terms of conducting electricity all of electricity is going to be flowing through the outside of the wire through through these bonds out here and of electricity is not throwing flowing through the middle of the wire service's unlike for example the copper wires that are used to wiring the walls of Hawaiian electrical outlet over there on the walls and on this property makes the outside of the wire superbly sensitive to tiny little perturbations on its surface and that's what we're going to do so here's way we do this students in the columns laboratory in my laboratory I start with silicon wafers that are about this big and we go to the engineering building across campus the students put on body suits and we build using photolithography circuits that look like this on their release contact pads to which we attach wires and then down here and you get down to the east in digital lectures that do not touch each other so this is an open circuit but on somewhere out here we sprinkle an IRA molybdenum catalysts that analyzes growth of 1 of these carbon nanotubes a single walled carbon nanotube across the wires to complete the circuit and I false colored red over here OK so now what we do
next is we turn
this wire into the world's tiniest microphone we turned it into a device called the field effect transistor it's not so how that works what matters is it's more or less the same as the microphone found myself pacing principle and next we're going to glue individual proteins directly onto the microphone and listened as they flap around runners if we had runners running through here you'd expect to hear the counting of the the right and you'd expect to be able to interpret that the noise of their feet to tell us something about the strike was not the accelerating whether or not the slowing down etc. will not be a funny he'll strike etc right makes sense so reducing thing but with proteins now I know but you're probably thinking proteins don't have noise but I am proteins all right body and I'm not hearing anything right now but the the truth is the new is fewer times it is so tiny that it's very hard to here but on moving charges do make noise and I'll give you 1 example of this if you're at the beach with the bonfire .period here little Corona Beach State Park and you have this big bonfire going the other way and kind of whips that the flames in the flames make this kind of neat whooshing noise and that sound of the flames moving is due to plasma in the flames that's charged ions in the flames that are moving around so charged functionalities make noises they get pushed around and in fact actually there's a speaker a loudspeaker is a really expensive stereo speakers on the order of like 20 thousand dollars pair from the better sound good at that price but it's actually based upon having a plasma that's moved around by little magnetic coil of testing you can actually hear charge clients moving around and that's certainly 1 legally with the protein so many show you what it looks like only the protein good
and this is and this is the schematic diagram over here we have the carbon nanotubes use protein gluten this protein is stripped out and familiar right everybody in this classroom and we have stripped out and conjugated to a tiny little thought of gold and that shown here because that little dots here that's the gold attaches strict and then the horizontal lines are the wires the carbon additives and the vertical are the electrodes over here and you could see we're getting 1 1 1 1 1 attachment is so that the the the breakthrough that that I came up with with our our co-workers and friends the graduate students was that we developed the way of attaching 1 and only 1 attachment each time to have narrative this means that that were isolating enzymes away from all the bodies which means that we can start looking at confirmations and intermediates OK this is all
along introduction really get back to the US and the UK kind in a moment before I do the need to set the stage and here's experiment again we have the electrodes cardinality but
it turns out that of course he can't run your cellphone and water so you can't run 1 of these tiny microphones in water right now I think I did this experiment last week I dropped my phone in a bucket of water matches my cat's water bowl and pulled up quickly enough but it definitely did cause some damage so electronics of water don't mix I don't think that surprises anyone in the classroom and and so for what we have to do is we have biology of course takes place in water so this creates a dichotomy and that's all that's what we do is we cover up all the electronics with a layer poly the factor this is shown during grade and then we blast the little tiny window using something called electron being that just opens up a little tiny region of the of and that's where we're going to do the experiment now all the images have been showing up till now electron micrograph using electron microscopy whenever they get really small as we start imaging individual molecules of proteins this is so small that you can't really see them very readily using electron microscopy except if you that trick that I share a previous library coatings of gold that was a stripped out and they so now that sees things we now are going to have to use atomic force microscopy worried now getting down to really tiny resolutions of 1 so so here's a looks like this is 1 enzyme attached to the commonality of and this is an the image atomic force microscopy image just showing the window region to showing the commonality that exposed this little blob right here is actually the enzyme attached it has the right dimensions for that 1 enzyme and now we turn on the microphone and it's lights camera action OK at that point ,comma were readied elicited pay
1 more image this is before and the next after and where it circled you can see very clearly the enzyme attached there was also was the questions so far had yes you go thanks for the help thanks to these other blogs the candlelight federal enzymes that we can get rid of it's actually enormously hard to be the sort of images it just turns out the proteins are kind of sticky and there might even be some salt somewhere out here so there's always some garbage stuff that we've been totally unable to get rid of despite a lot of work to took a lot of work to get images there is clear of the media in OK that's a good question but because of legal question actually takes about asking that things are not being on the study section that has such a case the questionnaires wouldn't enzyme in isolation from itself behave differently than enzyme that's next to its neighbors right in the same way the crowded field of runners was gonna run differently than in a solo run on we don't know 3rd I would like to think it's going to run the same but it is a legitimate caveat and I thank you for it I will think about that support thank you want to use the 1 you know and then you will yes so insurgents frequently are really set so it's true enzymes are really crowded conditions inside the cell but it's not like there's like a thousand enzymes that are all doing the same thing crowded together it's more like there's a couple when science ecology and then with hundreds of other molecules inside the cell others are not all doing the same thing so we can recreate that sort of thing we can wreak we can recreate the crowded conditions inside the cell and and I compare we haven't to the experiment but I'd love to do so that's OK with the back kind so
again this is pretty high he said it still has the 2 loves that I showed earlier years the Bigelow down here and here's the smaller Loeb up here and am somewhere close to the this upper alone Miriam together with the stability of former graduate student laboratory engineered a single system but the system of course has a sulfur functionality bile functionality and that allows us to attach site specifically the enzyme to the nanotube to this microphone down here through a pirate peso Pyrenees making yet another cameo in today's election and on as you might expect the hiring is going to pipe I stacked onto the Carver narrative OK because it's so hydrophobic it's looking for analogy to stick onto and it turns out that enzyme is very firmly held in place this is like a very special kind of molecular glue that sticks these 2 molecules together but notice that it's being held in non-covalent interactions and practice this thing is held in place for 10 12 hours we don't see it coming off it's really stuck in there very firmly again and again here's another Vietnam image and that little blob attach the cognitive is our inside case yet but over here Sergio yeah so we do and we have found this technique of atomic force microscopy before we get started with the experiment just to make sure that we have 1 attachment and we use a special technical illiquidity affirmed 1 if we see too attached we wash it away and start over if we see you 0 attached to a starter Rick detachment from the other questionable yeah OK so afterward so we don't have any more inside so we have like purified buffer there is no enzyme around said in a note your thinking you're wondering people want this club over here with the decides to give up the wander over here it turns out these blobs of pretty firmly stuff down on the surface and another thing is simply if another 1 attached we hear that 1 running alongside it at the right in the same way that 2 writers will make different sound 1 right you'd expect to hear a different rhythm and so we could detect that we don't see it for the questions the survey questions you guys get over here and so on we we only had 1 enzyme so we knew that we had to carry out the and the FMM which confirms that we have 1 that's attached to direct a question of activity OK I'm getting to that again what a general review from
and that he is impatient OK so he has said that the ghost exchange songs by some clout right there on the new Michigan and if you don't know want some classes but a great variety possibility of savvy and so we need the SoundCloud you know how like you said that you know so it is the of but also that comes with that it tells you about the loudness of the of the the thing I'm initially new images that are like that the that's like that we were going to be watching you know it's OK and that's shown here where all this is time on the x-axis and on the Y axis this is the current flowing through the narrative so that's going to be a noise OK and on enzyme by itself from waters around a little bit but for the most part it's totally quiet OK so if you don't feed the writer you know some oxygen or glucose you know some gel those some bars or whatever the writer doesn't start running again enzymes are like that to unless they get substrate this enzyme happens to be totally quiet some enzymes refined kind of randomly flutter around them in our technique will not that take now when we had a cheeky but we see a new let that appears to see this lower blip over here each 1 of these corresponds to the ATP downstate so we go from here I'm up here with the enzyme is open to down here words found and then back and we can measure how long the enzyme stands in this downstate and arrive at dissociation constant kg for this enzyme that this ATP interaction when we do that we find that that Katie corresponds to its measured ensemble kinetics that tells us that actually were seeing something very similar to what seemed more crowded cases OK next we wash away the ATP and then added in a peptide that's a peptide substrates this has the searing hydroxide that's going to be possible related by the gamma phosphate of ATP and again we see some intermediate confirmation as the enzyme goes from open to bounce again and again to see others like blitzed down here the enzyme points to its substrate is peptide substrates with greater affinity it grabs on tighter has been a lower paid eh federal so it may makes sense to obtain a check this out this is the really cool 1 this is now that the enzyme plus plus the peptide and now we see 3 levels OK so this is 1 2nd over here this is now two-tenths of a 2nd I'm zooming in and what we see is that these 3 levels correspond to 18 feet down and then can down which gives us a catalytically committed confirmation they so when enzyme starts working it goes between open intermediate and catalysis and I'm just going to call these 1 2 3 this is the waltz of PKA case so here's enzyme waltzing going along 1 2 3 1 2 3 1 2 1 2 1 2 1 2 3 1 2 1 2 1 2 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 4 that's an enzyme in action this is what the enzyme looks like as it goes about its business now which hugely inefficient is the chaos due member earlier I told you the perfect enzyme should have 0 chaos in this case we see the enzyme in real time in action being inefficient here it is being inefficient as a goes 1 2 1 2 1 2 this is it with the chaos track catalytic fish inefficiency is what lose the enzyme and makes it waste opportunities then enzyme is trying to make up its mind good a product that the substrate product substrate products substrate and that the lack of decision is what makes this enzyme inefficient as a catalyst there are other counts in kind pieces on such as kind cases involved in metabolism that are far far more efficient than this enzyme over here and if you're looking for something can I ask you just wait until after the class over I would know you're here for the class this OK no problem welcome there are so many like this is the enzyme in action and with the shows us is that the enzyme fluctuates speed enormously OK then this is kind of mind-boggling and I'm just going to tell a to it turns out that the enzymes the fluctuates from 2nd to 2nd by a factor of 100 cases this is like going out to the 73 out here the freeway out here and then you know pulling up alongside a Honda Civic and then suddenly the Honda Civic that goes from 55 miles an hour to 5 thousand 500 miles an hour and then back down to 50 miles an hour all in the course of a 2nd so these enzymes are wildly changing the speed their changing speeds much faster than any writer the changing speeds up to speeds the wildly there almost inconceivable to us humans and really that's the stuff of life it's essential that this enzyme is able to alter its speed in order for it to be regulated never earlier I talked about the regulation approaching tiny said that regulation is going to control its speed and in doing so that turns enzyme from being a wildly efficient catalysts to be a catalyst that's not even worthwhile that doesn't operate on a time scale that's useful for the cell and this is really the essence of how catalytic biology takes place in the questions of his year is the longer the on off on this OK so this is really a question of if we spent a lot of time looking for patterns in our data and looking for correlation between 1 step or another and we the insurer is pretty random and there is a small amount of memory effect in the sense that the enzyme if it hits number hits as intermediate state it's more likely to go down to 3 that is to go back to to 1 OK so that's actually affirmative Amica about the enzyme has evolved to do cat in preference to chaos erect
and let's talk about another Hydra let's talk about a different enzymes the 2nd and I'm honored to have I want to talk to you about today is laces I this is an example of a hideaways and remember moving down the still the top up here in most common enzymes this enzyme was discovered about 100 plus years ago and spin intensively studied this is X-ray crystal structure of our lights I'm and it that it was the very 1st inside X-ray crystal structure ever solved was this enzyme the active site up here but has been the Angstrom hinge motion and so this enzyme has kind a packed in like motion as a high to Wise's the Gallegos citic bonds of polysaccharides found on the cell surface of bacteria cells OK so here's a bacterial cell wall and enzyme is going to cut apart the people said bonds between each 1 of these quite convoy ladies and found on the cell wall OK knee-high chopping apart the polysaccharides in doing this this will basically burst apart the cell and in absences and signed it enzyme is basically given the chewing apart the bacteria and this has the effect of price of of killing the bacteria right you're breaking the cell walls they exploded etc. this enzyme is found in high concentrations in chicken eggs that's the headache whites over here and its present to prevent colonization by bacteria carrying might recall Avedon was isolated also from chicken egg whites so a biochemist and studying what makes up eggs so special is still still vessels for a very long time OK let's take a quick look at the bureau pushing mechanism for how this enzyme operates in this mechanism the enzyme goes through a through a covalent intermediate let's start over here so I'm zooming in and out to the polysaccharides region of the cell wall of the bacteria enzyme is going to cleave respondents indicated with an arrow and on example of a hydrolase meeting is going to introduce water across this bond so the 1st thing that the enzyme does is to work best but this this and acid till Grandma acid waiting OK so it's going to talk this carbohydrates from being a nice share confirmation to being a boat confirmation this is a crucial aspect for what makes enzymes such effective catalysts this enzyme is going to be catalyzing a reaction at thousand times more efficiently than if the reaction I just had to happen by itself that you because like 100 thousand times were officially and in order to do this the enzyme is going to be physically bending the substrate and by physically bending the substrate this helps the accelerate the reaction so far from here is pushed up into this both confirmation notice that the boat confirmation neatly sets up an Essen to attack by this carboxyl 8 other spotted gasses to attack backside displacement style this on the school glycoside this is crucial so the glycoside gets coordinated by 1 glutamic acid and then over here and a spot at the carboxyl at the carboxyl a from a nearby Espada gasses that attacks during a backside into to displacement and this coordinated like city bond then is a very effectively group because their secondary 0 highlighted in red over here it's electrons to a positively charged oxygen which is all too eager to accept those electrons OK so this gives us a covalent intermediate and this is another common way that enzymes accelerate reactions in this case were seeing former covalent intermediate and then this covalent intermediate gets Titleist so half of the polysaccharides plateau where the next 1 comes a water comes in and it's deep-rooted aided by the glutamic acid that good made up here and then this high July says the Esther bond between and this this mishap arrived at this polysaccharides and gasses and so on some notable features here were showing on show you an example of acid-base catalysis enzyme is simultaneously acting as both an acid over here and as a basis Over here in fact it's even wilder than that check this out it's the same functionality this glutamic acid that acts as both acid on the base and to me that's just elegant simplicity that makes enzymes so beguiling right and if we were in the chemical laboratory trying to make molecules you're using of glassware and such but we don't than a bunch of acid or dumping a bunch of base but you wouldn't add simultaneously both past and base because they're neutralize each other and on enzymes have evolved to be able to simultaneously catalyze states using both acid and base catalysis furthermore these enzymes of Alta form a covalent intermediate and sometimes referred to as a ping-pong mechanism that eventually it gives us back the high July like said bond paying really really beautiful this is what you learned in biochemistry classes and the problem with that is that it neglects enzyme dynamics which are really critical as the enzyme moves they can then helped to talk this confirmation of the rain into this vote confirmation in the absence of this movement it doesn't make sense really why it is that the enzyme is actually going to be talking this substrate right because the substrate lines in the chair conformation appear why should get pushed into this other confirmation unless the enzyme is doing the pushing and that in fact is what we see OK so the same
idea we're going to attach an enzyme to the carbon nanotube and then listen in as the enzyme works and what we
do that here's here's the reef of paper from 1 of about a year ago what we see is that again and then signed by itself is relatively quiet and then when we had this substrate the polysaccharides repetitive can showed on an earlier slide the costly that there's an immediate jump upward and then there's all this noise in here this is the enzyme chewing on a substrate we get the listener just like SoundCloud basically
OK but some control Simon showing you controls but these are everything in biology and this is the substrate by itself in red overlaid and then this is enzyme by itself again it's relatively flat and purple and then will we have enzyme plus substrate we see this motion where what seeing here His enzyme Open Closed Open pages have been the closed 1st opened because of the close of enclose them because this is it you know I'm a 3rd of a 2nd but we get to watch the Siemens and cranky over for a long period of time and again what we find is that the enzyme is highly variable it accelerates it slows down it speeds up its slows down and it accesses different conformations in fact access is at least 2 dramatically different speeds and you can actually see that in the 40 seconds of data over here and you see how the suspense region and analysts regional dense region against region corresponds to rapid switching the enzyme hasn't overdrive here that accusing Jews of eclipse skiers the 2nd year and it starts cranking along at a much faster speed and you can see that over here when the enzyme is going up because because of a possible a oppose it was like 300 times for 2nd worst over here is doing Open Closed Open Closed Open Closed like 50 times for 2nd so this is dramatic it's 6 times faster and what's crazy is that the enzyme does this all day long it's switches between first-year and second-year first-year second-year back and forth and a big mystery in the field what's up with second-year coach so to address that question
and I'm going to skip some stuff
To address that question together with collaborators we chemically synthesized a version of the polysaccharides that didn't have crossed like this is like 1 strand of the net that I showed you earlier seen polysaccharides now using chemical synthesis to access in the substrate and what
we find is that the enzyme has a different type of of of the activity I do attitudes more controls because I can't get away from this the crucial but these are mutant enzyme active sites to member earlier I showed you the carboxyl aids that are required for consigned to operate so we mutated those carboxyl at residues and ends I no longer works and therefore it never closes the other 1 of these enzymes mutations and traps the covalently bound form of the substrate that I showed earlier in the ping-pong mechanism and the enzyme never can hide your eyes back all the substrate and again it never gets back to close
OK so I get to finally tell you what the differences between first-year and second-year This is a day in the life of an enzyme but here's how it spends its time has so this is an enzyme print along happily from being fed either the linear substrate or the cross-link substrate in the case of getting the Crossley substrate it gets the high Joyce things about 50 per cent of the time but he billionaire substrate makers while against the behind eyes like a city bonds he Percent time and so and then this over here a 2nd year in blue that's non-productive but rapid chatter and over here and across late the Blue is much more appearance so in other words the cross-link substrate the substrate found on the surface of the bacteria cell corresponds to the 2nd year so what we think is happening is the enzyme is mulling across the surface will sell chewing contentedly Bond after bond after bond after Bonn happily high signal and then it hits 1 of these peptide cross links and gets done and when he gets stuck in its response is to start chattering away just it's what's years that just starts going 6 times faster than what we think is happening is that a contrarian sits along the peptide down to the parallel polysaccharides so in the same way that DNA is a pipeline direction Alania three-pronged originality polysaccharides have a directional is well and it turns out the surface the bacteria cell is a highway of parallel polysaccharides so they like comes along it's a cross-linked goes down zooms along its Crossley goes down zooms along down costs down across so the enzyme is doing is zigzagging across the surface of the cell as it choose apart the surface of the bacteria and in retrospect is totally makes sense because again the enzyme evolved to poke holes in bacteria and by doing a two-dimensional the surface the bacteria this makes the enzyme much more effective at killing its bacterial targets questions yeah so it is likely that the of yeah it's able to non-productive chatter that we think is moving along 1 of these peptide across lakes yeah Anthony until it finds a new Gallegos said bond and the goes to town again OK well that's let's move on I want talk to you about the enzymes have lots to talk about I want to touch it very
briefly about producers which likely common bonds and we've seen these we've seen examples of these in blood there's a whole cascade of producers better used to respond to damage blood vessels but with a series of bomb factory factors seven-day factor 10 etc. and produces where 1 cuts 1 and the next 1 cuts another that's on cuts another etc. and all the way down to the point where you get up production of vibrant which then could cross to form to replace the fix the damage appear case of of audit mechanism for blood clotting and unnaturally if you're missing any 1 of these producers or 1 of these producers happens to be mutated your big trouble you will your blood will not clot this happens in inbred families such as the royal families of Europe
and in the turn of the century this is the Azora Nicholas a 2nd whose wife Alexandra passed on the gene hemophilia but to the son Aleksey down here and again this is a mutation affected 9 or factory gate which are both excellent the genes that are found on the X chromosome so the passed along by the mother OK
apoptosis is also regulated by a series of producers apoptosis the cell suicide mechanism that we talked about earlier in this quarter and on each produce activates the next 1 so you have a pretty except here called the cast that cleaves the next cafes and wine which sank leaves this etc. so this guy cleaves the sky which pleases etc. But this
is a closer look at an example of a party is the police who want to show you is on 1 of my favorites it's isolated from 1 of my favorite fruits papayas and it's perhaps appropriately called tapping these isolated from papaya and it's an example of a 16 based creates is furthermore another example of a nucleophile based inside mechanism and I chose this 1 because 16 of course is the pre-eminent nucleophile as illustrated earlier today when we talked about engineering in a single sitting on the surface of the online as a way of attaching it to the to a specific spot on the carbon nanotube OK so
here's a here's the mechanism for how this enzyme works in practice it's actually a fairly complicated mechanism our science complex mechanisms in the sense that it's a concerted mechanism and specifically here the nuclear thought nucleophilic file functionality of the Sistine inactive side I don't think I
pointed out here but here's the Sistine that's the business end molecule in its active site
and in a concerted mechanism the Sistine is simultaneously deep related to attack nucleophilic leader Alan Bond and then the salmon bond Carbondale gets coordinated by In an acid residue that hovers above the and the of them in Bonn this happens in 1 fell swoop from nuclear file 2 protonation all at once and that is really the kind of concerted dance of that catalytic efficiency is yet another example of what makes enzymes so special was the fact that everything is kind of held together once but lowers the transition state energy rates and now you don't necessarily have to stabilize a protonated Carbondale inapposite instead you wait until more electrons appear up here on this oxygen that before it gets credited so that lowers the transition state energy for this transition state OK but there's other ways of depicting this as well I prefer this 1 concerted mechanism
and so all of these are OK GM escaped Syrian-based producers there similar to the existing spread
session showed earlier I'm a
citizen for areas of the well described as her quite a few others but like the kind raises the producers because they're involved in crucial process in the end human physiology such as blood clotting which you would not want to happen you know whether you know here and there and because because these are involved in such crucial processes and the enzymes themselves are tightly regulated oftentimes regulated by some loose in a pro enzyme that has to be clear where the terminology of Provo means a reaction takes place that then converts it into the active and functionality of the of the of the molecule sup example pro drugs are a precursor drugs that are then converted into the active drug by some enzymatic process and over here we see a pro enzyme that has eluded blocking access to the active site enzyme that gets cleaved and then that allows the pro and signed peace to get them to dissociate and turn on the enzyme so enzymes can be very readily
were inhibited you can do things like have transition state analogs we talked about transition state once before and here is the transition state for hydrolysis of an Alan Bond and here is a very effective phosphor Ahmad transition state notice that this also has the detector he took geometry of this transition state up here and if you do that you can actually very effectively inhibit this enzyme other types of inhibitors and fascinates down here boss rounds over here on the scale eyes are the dissociation constant for binding where it's KDE except it's for inhibiting finding and inhibiting the enzyme and against smaller numbers equals more potent enzymes and notice what a champion Foster and ideas it with a peak Imola near Peter Imola inhibitor
OK now there's a million other things I could talk to you about I'm going to pick them up next Thursday we come back will be finishing off Chapter 16 going on to Chapter 7 midterm will cover through today's lecture


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