Lecture 15: Enzyme Inhibition: Biochemistry I Fall Term, 2003

Biochemistry I Fall Term, 2003 October 1, 2003
Lecture 15: Enzyme Inhibition
Assigned reading in Campbell: Chapter 5.7 & 6.1-6.4
Key Terms:
Competitive inhibition Irreversible inhibition
Nonompetitive inhibition Protein phosphorylation
Feedback inhibition Zymogen
Allosteric effector
Links:
(I) Review Quiz on Lecture 15 concepts
(I) Data Analysis for Competitive Inhibition The handout shows enzyme inhibition curves.
(I) Structure of a Trypsin-Inhibitor Complex: "Must-viewing" of an enzyme-inhibitor
complex.
(S) ATCase Architecture: Chime images of T-State & R-State Aspartate Transcarbamoylase
with and without bound ATP (activator) and CTP (inhibitor).
(S) Graphing Quiz: Enzyme Inhibition Kinetics: Determine Vmax, KM and KI.
5.7 Inhibition of Enzymatic Reactions

Studies on Inhibitors are useful for:
1. Mechanistic studies to learn about how enzymes interact with their substrates.
2. Role of inhibitors in enzyme regulation.
3. Drugs if they inhibit aberrant or harmful biochemical reactions:

penicillin, ampicillin, et al.: interfere with the synthesis of bacterial cell walls.
methotrexate - anti-cancer drug that affects DNA metabolism in actively growing cells
viagra - interfers with nitric oxide breakdown (NO is a vasodilator)
4. Understanding the role of biological toxins.

Arsenate - mimics phosphate esters in enzyme reactions, but are easily hydrolyzed.
Amino acid analogs - useful herbicides (i.e. roundup)
Insecticides - chemicals targeted for the insect nervous system.
1
A. Competitive Inhibition
1. Inhibitor binds to the same site on the enzyme as the substrate.
2. Inhibitor only binds to the free enzyme.
3. Inhibitor is usually structurally very similar to the substrate.
Succinate/Malonate
ATP/AMP
The reaction scheme that corresponds to competitive inhibition is:
The inhibitor reduces the amount of E by the formation of EI complex. The inhibitor does not
affect the ES complex after it has formed. The dissociation constant for the inhibitor is KI =
[E][I]/[EI].
There are two anticipated consequences of this additional competive equilibrium:
Vmax is unchanged: At high levels of substrate all of the inhibitor is displaced by
substrate
KM is increased: Higher substrate concentrations are required to reach the maximal
velocity
Steady-state analysis of the effect of the inhibitor shows that KM is increased by a factor of
(1 + [I]/KI). The resulting form of the Michaelis-Menten equation is:
Measurements of complete saturation curves (i.e. vo versus [S]) at different inhibitor

concentrations can be used to obtain the dissociation constant for inhibitor binding. The
Lineweaver-Burk plots will show an unchanged Vmax and a slope that increases with inhibitor
concentration. (Figure 5.10).
Campbell's eqn. (5.18) for the double reciprocal plot shows that the slope of the lines will be:
Thus, KI can be determined by plotting the slope values vs. [I]. The resulting secondary plot or
"replot" will have a Y-axis intercept of KM/Vmax and a slope of KM/VmaxKI. The value of KI is the
intercept/slope of this replot.
2
The lecture handout, "Data Analysis for Competitive Inhibition", shows an example of the use
of replots to determine the KI values.
B. Noncompetitive Inhibition:
In this case the inhibitor binds to both E and ES. Both the slope (KM/Vmax), and the Y-intercept
(1/Vmax) of the Lineweaver-Burk plot increase (see figure 5.11). The KI ('s) are determined as
above by replotting the slope and intercept values vs. [I].
Vmax is decreased: At high levels of substrate the inhibitor is still bound.
KM is increased: Higher [S] is required to reach the lower maximal velocity. (For
"simple noncompetitive inhibition", KM is not changed, i.e. Fig. 5.11 in Campbell.)
C. Uncompetitive Inhibition (not mentioned in Campbell)
1. The inhibitor binds directly to the ES complex.

2. The inhibitor does not have to bind at the active site.
3. The inhibitor does not have to resemble the substrate (e.g. allosteric inhibitor).
Vmax is reduced: the amount of ESI formed depends on [I]. The solution to the steady-state
equation results in the (1 + [I]/KI) factor multiplying the [S] term in the denominator of the
Michaelis-Menten equation.
The slopes of the Lineweaver-Burk plot are unchanged (KM/VMAX), but the Y-intercept increases
by a factor of (1 + [I]/KI). The X-intercept shifts to the left by a factor (1 + [I]/KI).
Irreversible Inhibition
In contrast to the above types of reversible inhibition, where the effects on the enzyme reaction
depend on the concentration of an inhibitor (and its KI), there are many examples of compounds
that react chemically with residues in the enzyme active site. In these cases, enzyme activity is
destroyed. For example, the "nerve gas" Sarin reacts specifically with an active site Ser residue
on the enzyme, acetylcholinesterase. If acetlycholine cannot be hydrolyzed by this enzyme,
nerve signals cannot be passed across the synapses of the nervous system. On exposure to this
compound, death can result in minutes due to respiratory failure.
6.1 "The Michaelis-Menten equation does not describe the behavior of allosteric enzymes."
The above title statement, quoted from Campbell, is misleading. What is true is that the simplest
form of the Michaelis-Menten equation does not account for the higher than first order substrate
concentration dependence found in many allosteric enzymes. But this is the same as saying that
the Scatchard equation does not describe O2 binding by hemoglobin. In each case, the basic
concept of saturation (of an enzyme active site or a ligand binding site) is identical. In fact, the
3
cooperativity observed in allosteric enzymes is analyzed using the Hill equation and Hill plots to
obtain values for KM (or K0.5)and nH.
Allosteric enzymes are usually found at metabolic control points. Frequently, these are the first
steps in pathways. Regulation of the entire sequence of enzymatic steps is most efficiently
accomplished by turning on or turning off the pathway at the first step. When the control is
exerted by the end product of the pathway, the inhibition (or activation) is termed "feedback
regulation". An example to be considered later is the feedback inhibition by ATP of the
glycolytic pathway. Allosteric regulation always involves the binding of a metabolite to a site
separate from the enzyme active site. In this regard, the regulation is analogous to the allosteric
effects of BPG on O2 binding by hemoglobin.
(See the Lecture 15 web page for images of the ATCase T to R-state conformation change.)
6.2 Models for the behavior of allosteric enzymes

The concerted and sequential models for describing allosteric behavior are discussed in detail by
Campbell. Although the terminology has permeated the biochemical literature and is widely
(over-)used, bear in mind that neither model accurately describes any real enzyme-substrate or
protein-ligand interaction! Stated another way, it has not yet been possible to obtain data of
sufficient accuracy to distinguish between the two models. The most intensely studied system is
hemoglobin, where it now appears that a mixture of the two models may be the best (current)
description.
6.3 Phosphorylation of Specific Residues Regulates Enzyme Activity

We will consider specific examples of this type of regulation later in the course. For now,
simply remember that ATP is used to phosphorylate Ser, Thr, and Tyr side chains.
6.4 Zymogens
Campbell's introduction to the topic of proteolytic processing provides the basic concepts of a
frequently-observed regulatory mechanism in metabolism and in the differentiation and
development processes of organisms.
6.5 -6.8
We will return to these topics at a later date in the semester (after substitution and elimination
reactions have been treated in Organic Chemistry).
9.24.03

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Biochemistry I Fall Term, 2003 October 1, 2003

Lecture 15: Enzyme Inhibition

Assigned reading in Campbell: Chapter 5.7 & 6.1-6.4

Nonompetitive inhibition Protein phosphorylation

Feedback inhibition Zymogen

5.7 Inhibition of Enzymatic Reactions

2. Role of inhibitors in enzyme regulation.

3. Drugs if they inhibit aberrant or harmful biochemical reactions:

4. Understanding the role of biological toxins.

Measurements of complete saturation curves (i.e. vo versus [S]) at different inhibitor

C. Uncompetitive Inhibition (not mentioned in Campbell)

1. The inhibitor binds directly to the ES complex.

6.2 Models for the behavior of allosteric enzymes

6.3 Phosphorylation of Specific Residues Regulates Enzyme Activity

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