Module #6–Enzyme kinetics; inhibition and kinetic parameter determination methods; enzyme

catalytic mechanisms
UCLA, Spring Quarter 2019
o These are just some of the skills that you should be able to know by the end of this module
o Be able to holistically interpret an endothermic and an exothermic reaction profile curve
o Be able to interpret a Michaelis-Menten curve and be able to determine Km and Vmax from the curve
o Be able to understand the Km and substrate affinity relationship
o Be able to identify the five forms of enzymatic inhibition and be able to do so using the associated cartoon models
o Be able to identify the type of inhibition using a double–reciprocal plot (also known as a Lineweaver–Burk plot)
o Be able to assign inhibitor concentration to a series of linear Lineweaver–Burk plots
o Be able to calculate key kinetic parameters from double–reciprocal plots including Km, Vmax, turnover # and the
approximate time it takes to catalyze a single reaction
o Be able to interpret a catalytic mechanism scheme and decipher the type of mechanism that is being used by the enzyme

Slide # 1
Creating energy profiles of biological reactions o CENTRAL QUESTIONS

o Why did nature evolve enzymes?

Biological reactions cannot simply

rely/depend on spontaneity due to huge
EA
Reactions would literally take an eternity!

o What do enzymes afford?

• Enzymes lower EA barrier by offering

rate enhancement (KINETIC
advantage)

o Enzymes do NOT change G (i.e.-

reaction thermodynamics

Slide # 2
 How does an enzyme kinetics curve look like?

This is called a Michaelis-Menten curve

o Key things to go over:

o What is an enzyme?
o Protein that catalyzes reactions

o How do you determine Vmax?

o How do you determine Km?

o E + S → E–S → E + P o Km is an affinity measure value

o High Km=low affinity; Low Km= high affinity

Slide # 3
The concept and calculating/determining the turnover number (kcat)

The turnover number defines the number of substrate molecules formed into
products per unit of time

kcat = Vmax
[E]

o EXAMPLE with turnover # calculation and single reaction times

o What would be the kcat of an enzyme that has a Vmax of 60,000 M/s and an enzyme
concentration of 0.1 M?

o HIGHER the kcat, the FASTER the reaction

o LOWER the kcat, the SLOWER the reaction

o The reciprocal of turnover # can provide us with the time it takes to catalyze a single
reaction

Slide # 4
Inhibition of enzyme catalysis and the two broad categories
WHY evolve inhibitors for enzymes? An example of irreversible inhibition
o Enzyme function needs to be modulated in the cell (NATURAL REGULATION)
o Enzyme function needs to be halted or blocked ( DRUGS)

o Irreversible inhibition-modify the enzyme (active site) via covalent binding

(Also known as suicide inhibitors)

o Reversible inhibition (NON–COVALENT BINDING)

4 types of reversible inhibition

o Competitive-inhibitor binds to active site reversibly; prevents binding of substrate An example of the mechanism and mode of action
Example: o Competitive: similar structure of a common non–steroidal anti–inflammatory drug
to substrate (NSAID)

o Tamiflu binding to viral neuraminidase (enzyme breaks sialic acid on cell surface glycoprotein) o Aspirin acetylates a serine residue on
cyclooxygenase (COX–1)
o Non-competitive-binds to enzyme AWAY from active site; binding alters enzyme
shape, thus affecting catalysis o Please know that acetylserine
is formed
o Uncompetitive-binds ONLY to enzyme-substrate complex
o Leads to irreversible inhibition of enzyme
o Mixed inhibition-inhibitor binds to enzyme in presence or absence of substrate
However, it should be noted that sometimes there is a preference.
Slide # 5
o An introduction to modes of enzyme inhibition: four types of inhibitors (drugs) are known

o Inhibition is best determined through use of what are known as double-reciprocal plots
Lineweaver-Burk or a double-reciprocal plot of non-competitive inhibition

o NON–COMPETITIVE IHIBITION
o Inhibitor doesn’t interfere with
binding to substrate to active site
since the inhibitor doesn’t occupy same site
o I & S–dissimilar shapes
o Vmax altered
o Binding to inhibitor alters enzyme shape
affecting catalysis

o How to determine the Km value from plot?

o Extrapolate line(use a ruler!), make best estimate of x-intercept, solve
algebraically

Slide # 6
 COMPETITIVE IHIBITION Mixed inhibition Lineweaver Burk

Mixed inhibition
o Inhibitors CANNOT be catalyzed by the enzyme o Km increased (in presence of inhibitor)
o Vmax decreased

Go over Michaelis and Lineweaver-Burk cross-comparison plots

Slide # 7
 Lineweaver-Burk Plot for uncompetitive inhibition

o Uncompetitive inhibitor binds Uncompetitive inhibition

to a site other than the active o Km reduced–(in
site presence of inhibitor)
o Binding of inhibitor o Vmax decreased
stimulates
binding of substrate!
o This lowers the Km in the
presence of inhibitor

Summary table summarizing the effects of two key kinetic parameters in the presence of four distinct, reversible inhibitors

Type of inhibition Vmax with respect to Km. with respect to inhibitor

inhibitor
Competitive STAYS THE SAME INCREASED
Non–competitive DECREASED STAYS THE SAME
Uncompetitive DECREASED DECREASED
Mixed DECREASED INCREASED
Slide # 8
Cartoon models depicting the behavior of each inhibition system
Please be able to RECOGNIZE each mode of inhibition AND be able to draw it

NO INHIBITION IRREVERSIBLE INHIBITION COMPETITIVE INHIBITION

Normal substrate

Inhibitor competes with normal

substrate for binding to the active
site

Substrate binds normally o Imagine a deprotonated serine side chain in

covalent engagement with the inhibitor, I.

NON-COMPETITIVE INHIBITION UNCOMPETITIVE INHIBITION MIXED INHIBITION

Inhibitor can bind to

enzyme alone AWAY
from active site OR
inhibitor can target the
enzyme–substrate
complex.
Inhibitor binds to an allosteric In both cases, inhibitor
site AWAY from enzyme Inhibitor binds AWAY from active site. binds AWAY!
altering enzyme’s shape and function Binding of inhibitor stimulates enhanced
binding of substrate lowering Km Slide # 9
EXAMPLE:
o Constructing double–reciprocal plots from Michaelis–Menten kinetics data
o Obtaining kinetic parameters from these plots

Slide # 10
Step 1: Obtain reciprocal values AND ALWAYS label and show units (in bold black)

Slide # 11
o Go over each axis’s
approximate increment
value determination MIXED Inhibition

Lines intersect WAY OFF Y-axis

TWO KEY equations to know how to
use

–1 = x (x is this is the x-intercept value)

Km

1 = y (y is the y-intercept value)

Vmax

Slide # 12
THIS IS VERY IMPORTANT:
o Do NOT worry if your kinetic parameter
values are off from the problem set key!
o Your answers are being graded on
how well organized your response is
o Worry more about being thorough, neat, organized
rather than number matching!!
Please make a note of this, so we don’t have to needlessly
address this in the future.

Slide # 13
 • Go over how to estimate reaction
times from double-reciprocal plots
such as the model one shown below

– 1/Vo

1/[S]

• Go over a Michaelis-Menten plot

from the back (if we didn’t do already)

Slide # 14

o Do the Km and Vmax values make sense?

Practice example: Please do independently or we may do it in class

Slide # 15
Slide # 16
o Go over each axis’s
approximate increment
value determination

Slide # 17
 Calculation of Vmax

Slide # 18
Km for plus inhibitor = 10.0mM
o An introduction to the catalytic strategies used by enzymes
o Key question:
o What are these catalytic mechanisms that are used by enzymes
that helps them to achieve such high rates?
o In other words, what are the chemical schemes that enzymes
have been using for over a billion years?!

Enzymes largely rely on and use a combination of these four mechanisms:

Reactions take place within molecular pockets/grooves known as active sites (residues shown below)

General acid catalysis

CDESTY HKR side chain DONATES a proton

General base catalysis

CDESTY HKR side chain ACCEPTS a proton

Covalent catalysis
CDESTY HKR side chain make a COVALENT BOND with the substrate

Metal ion catalysis

A metal ion (zinc is very common) allows water to become a better nucleophile Slide # 19
o Example with interpreting a catalytic scheme of an actual enzyme
o KEY QUESTIONS
o What is the catalytic scheme adopted by Lys156 at the beginning (1) of the catalytic cycle? (General base catalysis)
o What are the two types of catalytic schemes adopted by Ser226 at the beginning (1–2) of the catalytic cycle?
(General acid catalysis AND covalent catalysis)
o What type of catalytic scheme is being utilized by Lys156 and Ser226 towards the end (4) of the catalytic cycle?

Substrate 1
2
FIRST Enzyme-linked tetrahedral covalent
intermediate
(ELTCI), circled in red
Product

Self-reminder:
Go over concept of
4 potential nucleophilic
maturity and immaturity
pairings in enzyme
3 active sites

SECOND Enzyme-linked tetrahedral covalent intermediate Slide # 20

ELTCI (circled in red)
 Another quick example
What type of catalytic mechanisms is being utilized?

a. With respect to His218, what is the catalytic scheme being utilized? Please briefly explain your response.

b. With respect to His56, what is the catalytic scheme being utilized? Please briefly explain your response.
the His56 residue lose or donate a proton.
B. With respect to His56, we are observing a general acid catalysis. The reason us that we are observing
bond in the reaction mechanism scheme
A. With respect to His218, it is covalent catalysis. The reason is that we are seeing a transient covalent
Answers:

Slide # 21
 An example of metal ion-based catalysis utilized by the enzyme, carbonic anhydrase, a metalloenzyme

o Main question: What is the function of the metal ion?

Overall mechanism of the enzyme What happens to the water molecule’s proton?

Slide # 22
 How are reaction mechanisms determined?–UNDERSTAND the concepts, no need to memorize the structures

o Researchers make use of compounds known as substrate analogs (resemble substrate but are NOT substrates)

o Substrate analogs are just a class of competitive inhibitors

o Why is this important?

Substrate analogs CANNOT be catalyzed by the enzyme, which helps to determine reaction mechanism

o Example: Proline racemase, an enzyme that converts L–proline to D–

proline

o Racemases or epimerases switch stereoisomeric conformations

Anytime a molecule
RESEMBLES a substrate, it is
likely a substrate analog and
thus it is likely a very effective
competitive inhibitor
Normal substrate Product

Slide # 23
Substrate analog, which is NOT catalyzed serve as COMPETITIVE INHIBITORS
Strongly binds to enzyme