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Lecture 25. Enzymes (second & final attempt)

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Lecture 25. Enzymes (second & final attempt)
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Lecture 25. Enzymes Pt. II
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UCI Chem 131C Thermodynamics and Chemical Dynamics (Spring 2012) Lec 25. Thermodynamics and Chemical Dynamics -- Enzymes Pt. II -- Instructor: Reginald Penner, Ph.D. Description: In Chemistry 131C, students will study how to calculate macroscopic chemical properties of systems. This course will build on the microscopic understanding (Chemical Physics) to reinforce and expand your understanding of the basic thermo-chemistry concepts from General Chemistry (Physical Chemistry.) We then go on to study how chemical reaction rates are measured and calculated from molecular properties. Topics covered include: Energy, entropy, and the thermodynamic potentials; Chemical equilibrium; and Chemical kinetics. Index of Topics: 0:00:06 Enzymes 0:12:36 The Michaelis-Menten Equation 0:20:27 Michaelis-Menten Kinetics 0:24:30 Ratio Between V and Vmax 0:25:34 Lineweaver-Burk Plot 0:32:56 Classifying Inhibitors
Beer enzymes
Beer Soma storage man
Beer Weinfehler Zeitverschiebung Meeting/Interview
physical chemist Beer Weinfehler Chemie chemical man
Weinfehler processes
grade Stickstofffixierung
Beer enzymes inhibitor kinetics Topic chemische Reaktion stuff man
enzymes mechanism rates substrates chemische Reaktion man
bond enzymes Lecture/Conference HPLC affinity active site substrates chemische Reaktion case complexes man
enzymes Bohr affinity capacities active site substrates chemische Reaktion chemical man
eds Geschwindigkeitskonstante mechanism rates chemische Reaktion complexes
enzymes function concentration Substituted substrates chemische Reaktion complexes form Knot
systems enzymes function rates Gamma kinetics substrates chemische Reaktion Knot chemical
enzymes flow concentration overexpression substrates complexes form Knot man
agricultural eds enzymes pharmakokinetischen concentration factors rates overexpression substrates radiation man
rates overexpression chemische Reaktion
enzymes important rates kinetics chemische Reaktion form
Alfred overexpression operation case man
Alfred systems rates chemische Reaktion Thorium
enzymes initial concentration Experiment indoor case Thorium man
Alfred enzymes conditions initial concentration substrates chemische Reaktion man
compliance enzymes Magnetometer concentration Gamma substrates Caffeine Mars chemische Reaktion Knot
type rates Bohr steps chemische Reaktion Bis (band) Knot man Tau protein shot substrates Caffeine form sequence
rates concentration chemische Reaktion case Bis (band) man Alfred gap enzymes initial Lecture/Conference overexpression substrates Caffeine
enzymes concentration Dielectric spectroscopy substrates Caffeine complexes chemische Reaktion Bis (band)
enzymes flow Geschwindigkeitskonstante concentration rates steps Caffeine chemische Reaktion case
Insulinkomabehandlung enzymes flow Meeting/Interview Dielectric spectroscopy substrates Caffeine chemische Reaktion man
Clenbuterol substrates function concentration rates overexpression substrates Caffeine chemische Reaktion man
enzymes substrates function concentration rates chemische Reaktion man
substrates rates Bohr substrates chemische Reaktion man
Zellzyklus enzymes substrates rates overexpression kinetics chemische Reaktion man
Zellzyklus Geschwindigkeitskonstante substrates chemische Reaktion man
Revenue Zellzyklus enzymes substrates Brown adipose tissue rates concentration chemische Reaktion Medroxyprogesterone
hard Revenue Zellzyklus conditions Blood vessel Kath Brown adipose tissue rates Wasserscheide Zigarre chemische Reaktion
Grease interceptor enzymes function rates concentration Insulin solutions chemische Reaktion slides buffer
Grease interceptor solid-phase extraction initial concentration rate Klinischer Tod substrates man
emission rates concentration Bohr substrates chemische Reaktion man
concentration rates Sheet chemische Reaktion Stöße man enzymes initial race Magnetometer function Klinischer Tod substrates
Trihalomethane Manganese
match Kunststoffe Katalase man
Grease interceptor enzymes kinetics man
molecule type enzymes inhibition Magnetometer Lecture/Conference concentration kinetics inhibitor substrates chemische Reaktion man
enzymes Bohr active site bind inhibitor substrates stuff man
van enzymes Magnetometer Lecture/Conference active site inhibitor substrates complexes man
enzymes enzymes BET theory inhibitor bind active site inhibitor substrates complexes
initial substrates concentration inhibitor inhibitor substrates chemische Reaktion
Seltenerdmineralien enzymes substrates enzymes concentration Gamma Macintosh inhibitor substrates inhibitor chemische Reaktion man
Magnetometer substrates concentration inhibitor inhibitor substrates slides
inhibitor inhibitor man
enzymes Lecture/Conference concentration plant inhibitor active site inhibitor case man
Grease interceptor enzymes concentration inhibitor substrates complexes case man
enzymes active site inhibitor substrates case chemische Reaktion complexes man
Grease interceptor Digital elevation model Lecture/Conference Magma man
Digital elevation model enzymes initial concentration Magma substrates derivatives biochemistry man
type enzymes mechanism substrates inhibitor complexes chemische Reaktion form man
conformation analogue inhibition enzymes Zigarre Methylmalonyl-CoA-Mutase inhibitor substrates complexes man
Alpha analogue enzymes concentration inhibitor substrates equilibrium constant man complexes man
Alpha inhibition sodium hydride inhibition enzymes inhibitor case
Alpha type inhibition Lecture/Conference mode inhibitor Dielectric spectroscopy inhibitor case
Alpha eds mode active site inhibitor Sand man
rates inhibitor case chemische Reaktion slides
Wasserstoff enzymes polypeptide chain Bernsteinsäure chemische Reaktion membrane
marches substrates Double bond carbon Säuren food Wasserstoff Säuren active site inhibitor form stuff Fumarsäure
OK for a given signs another try today but so there is a mistake in the way
I calculated the Howard told my doing and the mistake is that if you missed equated not a together 0 but a few minutes to quit was then it was calculating your how my doing score incorrectly so few of you pointed this out to me and I fixed it and so I posted a new all my doing score on Friday it's
separate from the earlier 1 in so if you missed
quiz your home I doing scored should be higher than it was on Thursday righteous check that if you would like to thank goodness of a few of you went to the trouble that check your all my doing store and found this OK
quit 7 I think are already posted all right so that should be
all of the quizzes for the quarter you should see all across all this cost them the posted as
well on the results page Chris 6 stores have been updated we made
an error in the way we agreed that that Excuse me completely my fault there was an error in notation that we used on the quiz and so Jean-Marc went through and regrade all
of these and repulsed them so that crisscross the up their request 6 are updated if you want double
check your quiz score that would be a good thing to do this book the
electronic evaluations that turned out last week and a few of you have to work them out for me but so far just 16 per cent of you I need and there's no way
I can force you to do this but I want to ask you if you would please take the course evaluations let me explain the let me explain the situation is faults but I teach general chemistry analytical chemistry physical chemistry when the teach general
chemistry evaluations from the students there are
almost useless the reason is
it's not really their fault think the students right here they don't know if the drill instructors
a good guy bad guy on intermediate guy they just know they can't hold their feet
up for 30 seconds but they
have no experience with drill instructors ended so when I get feedback from them you know I like and feel like I've done the best job in the world of teaching his class and I will get all of this extraneous feedback you know you answers something wrong your you
guys are like these guys right here right you're grizzled
veterans most of your graduating in for weeks and he's seen everything you've seen bad teaching using good
teaching seen intermediate teaching you hear calibrated you know what exactly
what the issues exist with the class right and owners issues with this class and would really help me out if you went through did these evaluations and not only just scoring them because I know that the easiest thing possesses radio buttons but if you actually wrote some comments to help us make the course better I read every 1 of these comments I get a printout of a list of all the comments I read those old and I don't get to see them and for about 2 weeks because they don't allow me to see what your comments are before issue the final grades but after final grades a week after that they let me see with your comments are OK so please if you were to dine on takes time pride takes about 10 minutes 15 minutes to do this I'd appreciate it far so we're
going to talk about enzyme kinetics turns out all of this stuff is in Chapter 21 of
your book write a new chapter that's called catalysis so what
we want to do and once again all we've been doing this rebound
cherry-picking certain topics From stat thermal kinetics there are the most important topics I think so this is 1 of those topics Clinton the basic
ideas we want understand how enzymes catalyze reactions no we're not really learning how and signs do this work studying
the phenomenology of enzyme catalysis in other words were looking at the rates of enzyme reactions were trying to understand how we can break the mechanism of enzyme substrate catalysis down right here turn it into a modular thing that we can assign Constance to and make measurements on and compare enzymes against 1 another and so forth
right we're trying to really understand the phenomenology of enzymes substrate catalysis so we've got an enzyme we've got a substrate but when the substrate Dotson the enzyme what this schematic diagram years trying to depict is that there
is a recognition event that has a worker In
other words the enzymes not going to catalyzed this reaction whatever it is for any
substrates wait there has to
be a recognition of the substrate by the enzyme at the active site in order for the substrate to darken once the stocking Kurds
then the enzymatic rule in somatic reaction can proceed In this case it
looks like some sort of bond breaking reaction occurs OK so this enzyme substrate complex is meant to recommend wrecked represent this entity here all right and then products are produced and released from the enzyme at that point once this reaction occurs the affinity of the products for the enzyme
is lower In the affinity of the substrate for the enzyme if that was not true the products
would just stay bound the enzyme and would be game over OK so the enzyme has to release the products once the formed in the active site if that didn't happen the enzyme would be pretty useless wooden so that's what this
event is depicting here OK so we want to work out some
equations that are specific to enzyme catalysis that help us to understand these reactions the batteries in my laser pointer are dying so many use it sparingly but hopefully it'll
get us through this lecture here's the mechanism I shot on a previous life here's the rate of the reaction
unjust depicting here the rate at which he is formed or I think you can see that that's it to you the molecular reactions from the enzyme substrate complex with the rate constant K 2 we're going to apply the
steady-state approximation again right it's another example of that and to do that we
set the time rate of change of the intermediate EDS equal to 0 and so I think you can see there's a rate at which he has builds up in 2 ways at which has is consumed and
so but that equation with a written down earlier because it's just the steady-state approximation and now we have to say some things that are specific to enzyme reactions we're going to make some substitutions into this equation and 1 of the substitutions who wanna make
it is for the enzyme
concentration because we don't know a
priority what it is is a function of all right presumably the enzymes going to get bound form the enzyme substrate complex and the free enzyme concentration is going to go down in the enzyme substrate concentration is going to go up presumably that that's what has to happen and so we don't know
what that is but he is but we don't know how much
total enzymes we've got but at least afford studying this reaction in the laboratory we added a certain amount of enzyme at the beginning of the experiment to study the enzyme kinetics that we're trying to Starting in the letter right now in a natural system no if they're ourselves around and we've got some extra act from a liver and some enzymatic chemistry is going on we don't know anything right we can't study enzyme kinetics under those conditions in general but the study enzyme kinetics we gotta take the enzyme controlled the pH but some substrate in contact with it and measure the reaction rate as a function of time somehow we've got to do that OK so
presumably we know that the 0 at least doing the
experiment in the lab we know it so the total enzyme
can only exist in 2 forms Free enzymes an enzyme substrate complex and so now I can solve for the free enzyme in terms of being told concentration of enzyme and of course this is
equal to the total concentration minus the enzyme substrate complex so now I can plug that into this expression bow here's my new studies approximation all
right and when I distributors over these 2 terms I am now going to get 4 terms instead of 3 1 2 3 4 terms and I can move the 1 generation term over to the left hand side put all the minus signs on the right side alright so all of that has got to be equal to that of this is equal to 0 and I just saw this for
EDS and that's easy to do because I got BS BS BS factor that out but and this is the expression that I get old I want to remember that this he has concentration
that I'm using now refers to the steady state and substrate ,comma concentration right we're assuming that the steady state approximation is correct
so this is the equation I I get for that an earlier we said that the rate at which he has formed a statistical decay 2 times the enzyme substrate concentration and so now if I just plugged yes into here I get this equation right here looks
like a mess but OK but I'm going to
divide the numerator and denominator by once OK one's going to go away here In K 1 is going to go away here and K-1 is going to end up in the denominator here right so this is the no expression for the rate of the reaction that I get still subject to the state approximation and now I'm a role all these constants up and call on the M K
big M write all those guys
together are going to be the Mikhail constant in this equation Is the Mikhail Menton equation it is the
most important equations and enzyme catalysis OK but it's moderately useless In other words we derive this
equation it's very important it contains all the kinetic rate information for our
and enzymatic reactions but we can't extract information from this equation very
easily in the form that it said ironically it's the most important equation enzyme kinetics but we can't really use it
but we need to do some more work what we need to do will 1st of all let's
think about this equation and
see words telling us notice that in the denominator there's an addition operations What is that there's going to be limiting
cases right if there's an addition operation In the expression for the raid right there's
going to be limiting cases worldwide because 1 half of this addition operation could be large compared to the other half or vice-versa OK so we've got in addition operation denominator 1 the corresponding limiting cases will it take to it member teachers part
of KTM the taped to his back
How big big compared to the minus 1 and 2 over K-1 if that's big compared to last there this thing simplifies quite a bit notice 52 is large minus 1 can be neglected and after being neglected scientist at K 2 over K-1 and K-2 is going to cancel and sold the rate of the reaction is going to simplify vary
substantially but the rate of
reaction system became 1 times the 0 the
initial concentration of enzyme times the substrate concentration as is the good
yes there were near that should always be true the key
to his lodged if the steady-state approximation is correct the answer is yes ah
but we're going to abuse this equation on a routine basis all right so this limiting case is not always going to be observed all well and it was used if
it turns out it is useful and the reason is that we're
going to apply it to the initial In other words were going
to measure of away at the beginning of the enzyme substrate reaction using an initial concentration of substrate and an initial concentration of enzyme and under those conditions this equations gonna work pretty well OK so what is
this if K 2 is big art we derive a simplified equation but conceptually what is that means but is Ermina K 2 was
big what it means is that
the reaction doesn't even know yes exists right of K 2 as big as soon as he has formed boom it reacts immediately right so it's
concentration is very low which is good that's in compliance with what the steady-state approximation is assuming right the enzyme substrate concentrations gonna be quite I constant because it's very long but this
also means that is approximately equal the E 0 because the Athens occurred approximately 0 right and so essentially what
this means is that the formation of the EDS in this 1st reaction here is the rate-limiting step In this
sequence of reactions right at the
rate at which the DSS Form Kansas soon as the STS is formed a boom it
reacts like a shot but might
notice also that the reactions first-order and substrate right in this limit of hiking to the
reaction is 1st substrate will come back to that 1 of us is
very bright investors big then I can neglect this whole diner parentheses here right and I just end up with this expression right here in S is going to cancel and so the reaction rate is just in that case just given by this expression here it doesn't even
depend on us not only that it doesn't even depend on
time but it only depends on the initial enzyme concentration right in the limit of large
gaps large concentrations of substrate the reaction rate is constant but you don't see any change in rates so conceptually what we expect to see that low substrate concentration what would OK what is this means
what's happening the means in this limit when as is big
all of the enzymes tied up enzyme substrate complex right we've get very high concentration of S we've driven this reaction forward because its first-order
analysis and we've basically saturated Orleans I'm all the enzymes got a substrate on it but there's so much substrate around that this
reaction has been Germany so far to the right based on which at least principle that we've tied up all of the enzyme is and that some Street complex beans and substrate complex concentrations approximately equal to the
total enzyme concentration OK this means
that the reaction of EDS is real this 2nd step is now rate Ltd OK and that's what it looks like right amusing that rate constant for the total reaction rate zeros just equally yes all right so in this limit the reaction doesn't care about the substrate concentration what came minus 1 as big not very interesting it happens reaction will be slow
not an interesting limiting case but 1 that could happen right minus 1 could be large
compared to K 2 and then came as 1 of a 1 could be large compared asked What if that's true the
equilibrium lies far Towards the substrates equilibrium
lies way over here right and it's a bad enzyme
it's not a very good enzyme enzymes not recognize recognizing the substrate maybe the substrate is not the normal substrate for that enzyme reaction rich just slow OK and sent the substrate is a 1 a dock with the enzyme
performance and I'm substrate complex OK whatever is small what happens at low
concentrations of substrate
check it out all right here's the Michaelis-Menten equation right if I make s smaller disappears from the denominator up with this expression right here the reaction is first-order ns OK so we said when is
bigger reaction rate becomes constant when asked slow the reaction rate is first-order NAS OK
so we know what
this reaction is going to do is a function of yes by it's going to
be first-order at low as so this is a plot of the reaction rate worse the concentration of guests at small as we see first-order kinetics for s in other words the reaction rate goes up
Lee nearly as a function of the concentration of as I think you
can see this looks like a straight line down here right but as
as its larger starts to and at
high concentrations of as it becomes concentration
independent right that
limit is the maximum reaction rates the
max that's a good decade 2 times total concentration of enzyme that I started out with in this reaction right so this is what
any enzyme will do as a function of the substrate concentration by that what
this equation predicts right and it's also what's experimentally observed now map what this among other things that are indicated here that I will
explain to you the liver the
largest we obtain the maximum rate to the max I show that you but here's the V max and we're not quite there yet we would have to go to a higher and higher and higher concentrations of substrate but eventually reaction rate will asymptotic Lee approached this dashed line which represents the map sets the
maximum reaction rate for the enzyme that a reaction rate that is characterized by the fact that the enzyme every enzymes that a substrate stuck on it so at that point the reaction can go any faster but that OK so K
2 times use of Emacs yes of now
there's lots of things in enzyme kinetics the confusing but here's 1 of them alright so let's get rid of 1 thing that's confusing here the next every
0 is given by this expression right here but we also call the next over E 0 the turnover number I think you could see in the next over here he zeroes obviously equally Kato but
1 of the units of K 2 be
seconds to the minus-1 why isn't he s reacts to get products right but so tight you know molecular reactions so the rate constants can have units of 1 over 2nd OK and so
that's OK so the turnover number is going to have units
up per 2nd reactions per 2nd it is what it represents OK so the
necessary 0 unadjusted so if we work out the units this just shows that the maximum old Moeller per 2nd if you will that's the
reaction rate the
concentration of enzyme is smaller so the units of this quotient here are won over seconds "quotation mark we're gonna call that K catalysis or K 2 or the turnover number 3 of those things are the same but some said the
turnover number the just talk about K 2 summonses Kate can the distract McKay to summon says just talk about the
turnover number because they're all the
same thing they all have units of 1 over seconds that confusing why have 3 names for the same thing I don't know but look at
now let's take the ratio between the reaction rate not just gonna call the reaction rate VAT remember
I was calling it deep PDT no different and discolored V the velocity
divided by the maximum Rabin K to disease 0 5 divide these 2 things obviously that's going to cancel this I'm just left with passed over as was carrying out an abiding the reciprocal of that I did this and if I divide by the max and cancel these vessels over here I get this which is the same as this so all we've
done this to take the Michaelis-Menten equation and do some more algebra on it To get an equation that is not held the
line we were Burke equation it
should be called the line Weaver Burke equation but it's not but
if you make applied using this equation is Colline Weaver plot make plot take 1
over s that's going to be your vertical axis but no that's
supposed to be excellent schools to be so that's the vertical axis horizontal axis rather the sludge to Taipo this should be y 1 over the velocity is the vertical axis right Lopez this interceptors that think USA slide for 3
years and never notice right to be clear we're talking about the initial substrate concentration in the and Nichelle velocities all right so you take your enzyme solution in your pH controlled buffer right you add some substrate and you measure usually spectroscopic Lee how fast the products are formed as a function of time in Newark's you arm extrapolated 0 times by any measure the reaction rate at time 0 course you're measuring initially the reaction rate over a range of times where you extrapolate to 0 to get the initial raid
we call that the 0 right and that applies of course to the initial substrate concentration that's usually what were plotting line Weaver Burke what here's what looks like right 1 arrests horizontal axis 1 over the vertical axis notice it says of the 0
that means the initial velocity right this should really say S 0 that's the initial substrate concentration right we should
get a straight line from that the slope of a line is going to be Qayum over B max the intercept on the Y axis going be 1 over the Maximina step the x-axis is going to be minus 1 over M yes check it out if I take 1 over the annihilated 0 and I saw all 4 white 1 Alvarez is going to be at 1 over the next 2 0 0 idea minus 1 of K M right from
the Nightline Weaver Berg
equation OK so this is an extremely useful equation and Micaela cement
equation now not so much right now useful except that I can derive the line not Weaver work
equation from it and that's really useful here a problem from last year's final exam by
the following results are achieved so to soon substrate concentration reaction rate should say the S 0 emissions saved me 0 which
substrate concentration is it is a constant during this whole reaction not necessarily right going can be consumed as a function of time right Is there room
reaction rate and the constant is a function of time during this reaction not
necessarily all right we're really
talking about the initial substrate concentration initial velocity here some data tell me about this
enzyme I want to know everything about it kinetic behavior
right while we can get everything in other words we can get K M they wouldn't get a 0 right now we can get the max by making aligned with the Burke plot so to make collide with work but we don't want asked me what 1 over as we don't want the what 1 over the I want a plot 1 over the race's 1 of arrests here's a piece a graph paper that you're going to have on your answer sheet right here the
data points you're gonna plot you're going to drop its access is you know you don't want it here are you making line we will work a lot you wanted in the middle somewhere because you want thanks to at think about 1 over the Mexico 0 this
dashed line here is meant to represent the maximum possible errors that any student that possibly make plotting these data points so we
can greater OK no it's helpful to you is to help a straight edge so that we can
plot these data points here and then trust
straight line through right so maybe you can use the edge of your notebook but if I was you I would buy 1 of those cheap plastic rollers are about that long that's very handy for this purpose a straight edge happened OK so
now that I've got this I can determine the match K M K cat all of these things
everything that you can possibly determine in terms of the observational kinetics of this enzyme we can extract from line Weaver Burke plot OK getting the right answer starts with getting the right line Weaver Burke plot and the right intercepts the right slope OK you with me so far this
story under Chapter 21 now How many people heard everything I said so far in another class for now what could
possibly mess this up well inhibitor write a
molecule that inhibits this reaction
could mess it up right and inhibitors are extremely important in enzyme
kinetics and this is the beauty of the line
Weaver Burke plot right how the line Weaver Burke plot changes with and without inhibitor is diagnostic of how howled the inhibitor is acting on the inside it's a thing of beauty right you can measure the line Weaver Burke plot without the inhibitor add the inhibitor and look what happens to the line with Burke plot and little more inhibitor a little more inhibitor all right of course you have to measure it is the initial velocity of the reaction at a series of substrate concentration right for added inhibitor all right but once you've done that you can determine exactly what type of enzyme inhibition is occurring there's 3 the innovation
could be competitive and noncompetitive or uncompetitive Is this another
example of confusing and sign terminology yeah not at all and are
not the same but I don't
know who came up with this stuff OK so what's the difference it's very
simple In competitive in an enzyme in addition the inhibitor is finding at the same active site that the substrate once the buying right if the inhibitors in substrate can bind its blocking it but that's competitive inhibition the word perfectly matches what's happening In
noncompetitive innovation the inhibitor is
binding away from the active site any combined either to the free enzyme orderly and then substrate complex but it's binding away from the active site it's not binding at the active site so the innovation is not competitive and finally in not uncompetitive
inhibition the inhibitors
binding away from the active site but only at the enzyme substrate complex right so there's a subtle difference right
noncompetitive and competitive and noncompetitive the
inhibitor combined either to the free enzyme within them substrate complex noncompetitive in uncompetitive it can only bind to the 3 complex see the difference Free and Xiamen's enzymes at the complex that not on and then substrate complex only bets on good luck it's hard to remember right here's
a to here's the inhibitor here's the substrate the inhibitors blocking the active site that this piece of the pie here but once the innovators in there the substrate camp I'm
obviously the reaction slows down but in a very characteristic way right
it slows down and so
here's what the Michaelis-Menten data looks like right initial
velocity initial substrate concentration right here's what happens in the absence of unit inhibitors now I had the inhibitor the whole curve shift to the right but if I make a substrate concentration high enough I get the same the max what's happening I'm out competing the inhibitor
I've got a certain inhibitor concentration in a fire at enough substrate eventually this guy
wins over this guy all of the enzymes get tied up by substrate if there's a thousand substrates for every I
think you'll agree the the substrate to start to win and that
limits I get the same the Mac's where the enzyme is
maxing out during a reaction that I care about the inhibitors having a relatively small effect on them OK so if it's a competitive inhibition situation you can always out-compete the inhibitor by making the substrate concentration the Norman In that limit you get the same the
that's what does that mean so so let me point out
that in competitive inhibition you see how this is half the next year this -dash line right here what happens that the Max wires
that interest in what turns out to have been Max if you do the math you can use equal substrate concentration a put on the
slides you can check it out later on all right here's the substrate
concentration that's equal the carry at the Max over to
parts of your just looking at your ID that you have made a line Weaver Burke plot yet you can look at
this plot right here you can go to have the Max that the Qayyum right there and so you can see the effect of the inhibitor is to increase
all right but it doesn't affect the next 1
influences that have on our line over plot this is 1 of a rematch it does not change for competitive innovation so if I had anything here is no innovation now I add inhibitor now I had some more no I had some more by a series of straight lines that have the same interests out but different KTM His KTM 1 overcame is down your minus 1 over KTM 1 over the next right and I can look at the
slimy Verba plants a boat it's competitive inhibition obviously right same
intercept difference and the slope is changing in this it's getting the slope is getting larger as I increased inhibitor concentration
what about noncompetitive while in this limit here's the inhibitor is finding away from the active site the see that it's binding a combined in this case from showing here it is binding to free enzyme but can also buy To the
enzyme substrate complex part of it's noncompetitive inhibition
In that limits what happens His caveat is unaffected but V Max hits reduced I said this is 1 of the rematch remember the
Somerset 1 Overview Max so as I increase the inhibitor concentration Qayum doesn't change but the interception just the slope gets bigger I can look at this I can easily tell the difference between these 2 cases right
here French disguised as well as preserving this intercept guys preserving the center said
writes line Weaver plot allows us to diagnose immediately which of these 2 cases is operating in me I now when I had inhibitor the
reaction is slowing down I can
see that right but I want to understand where the inhibitor how the inhibitors acting on the enzyme is a plug-in up the active site is a binding away from the active site is doing it for the enzyme substrate complex only or for the free enzyme as well but I can tell that despite being a
few experiments finally uncompetitive amazingly give us a fair
case that we can easily tell the difference in noncompetitive
this slot which is clear
Max turns out that's
slope is preserved and I get a different interceptor 1 Overby Max different interceptor won over K M but the ratio stays the same the amazing so I
can classify I'm willing to do
the work because there's a non-trivial amount work here I mean you've gotta do 4 or
5 experiments to map out every 1 of these lines with
different initial substrate concentrations different initial enzyme concentrations given initial velocities now but if you
buy this book which is the
bible of biochemistry
even if it does not contain what I'm about to show you which is the
mathematical derivation of where the straight lines come from it's not in your book it's not even in injured its value
added that you can get very easily taken anywhere else what are you ready for this and I'm not going laboriously and do all this math and is going to click through the slides and show you because it's a thing of beauty
here is the mechanism that we've been talking about so far right reversible formation the enzyme substrate complex reaction the enzyme substrate complex to get products right now we're going to add to other possibilities the enzyme can reversible lane form a complex with the inhibitor that's fine by any enzyme substrate complex
conformity a complex with the inhibitor as well the exodus to new possibilities that we have to consider these 2 new
possibilities encompass all of the things that can happen is that all 3 types of
enzyme inhibition competitive and noncompetitive and uncompetitive OK and all
we do is we we write a conservation of mass equipped equation the total enzyme that I'm putting in my bigger asked existed 1 of 4 states free enzyme and then substrate complex enzyme inhibitor complex enzymes substrate inhibitor
complex because only 4 possibilities right but now I do the math on this and this
is what that looks like I have to create some definitions amended derive something called a minute defined rather something
called Alpha which is 1 plus
the inhibitor concentration over Big K 1 with the K-1 that right the equilibrium constant for the inhibitor interacting with free enzyme that equilibrium
constant what's K 1 prime that's the equilibrium constant for the enzyme substrate complex that's going to be a private 1 plus that over OK and so I do man and I do a little algebra we can write equations it doesn't take that long
before we get to your equation 21 . 8 dB all right here still non-aligned Weaver work equation here's the Non-Aligned Weaver Burke equation for enzyme inhibitors right
check this out and got Alpha Prime instead of the 1 I've got a case times the Michaelis-Menten constant all right instead of K rights of just inserted Alpha Prime here and l the here and now I've got an equation that is completely general for all
types of enzyme inhibition all right this equation explains both 3 behaviors that we just talked about but how does it do that 1st of all on notice
that if Kerry Alpha sorry if Alpha Prime is 1 and Alpha is 1 then the inhibitors exerting no effect I just get the
non-alignment line Wieberg equation right that this case
Alpha equals 1 of a private was 1 zilch nothing
happened all right for competitve
innovation Alpha is greater than 1 Alpha Prime equals 1 max is not change became gets bigger this tables
a thing of beauty it's also not found 1 inches
noncompetitive inhibition inhibitor binds to bowl EDS in iii away from the active site yes yes yes both Alvin crime are
greater than 1 the next it's
smaller K. Anderson change that's what that actually means but these 2 offer means and finally uncompetitive innovation Alford doesn't change but Alpha Prime is greater than 1 both of these 2 things are smaller but
the ratio is preserved so the slopes stays the same bernanke search for on competitive innovation these are those
3 cases I just showed you I just put the slide in your industry can look at it next to theirs yes so 1 thing that should be obvious Is that for
competitive inhibition rate the inhibitor looks like the substrate if it doesn't it's not going to inhibit the reaction this is the
classic competitive inhibition reaction suddenly dehydrated Denise D hydrogenated sought right this is the mile
Cotterell membrane here's what that enzyme looks like it's got a trans membrane component that plugs into the middle Cotterill membrane is delivered by layer right we've got the but I often feel a cold peptide regions that are very hydrophobic that like to insert into the lipid violators right and then we got this more globular up extracellular part here's the reaction here's the Sox nature that's this guy right here is the full that's the product of the reaction but that's the
accident after gives the hydrogenated Saxony's CDU hydrogenation
sucks in each the hydrogenation will form food Mourad form this double bonds all right the classic competitive inhibitor is melodic acid look at this and look at this alright if I rotate about that single-mindedly you're gonna agree that this looks a lot like that there's an extra carbon here alright but melodic acid inhibits sex the
hydrogenated sucks 8 the hydrogenated inhibits the heck out of it very
wise because this is the active site gobbled the stuff up it looks just like this it's very similar in
size OK the official are there any questions about this because I Wednesday talk about some notes OK look at Chapter 21 and then CBS questions about this stuff