林重銘老師生物化學 ( 一 ) 講 110-01 CH13-2 V1

義
第十三章第二部分

Chapter 13
Enzyme—Kinetics and Specificity

Modified by Chung-Ming Lin

Department of Biotechnology
Ming Chuan University

AA509
EXT 3955
Enzyme Unit Are Used to Define the Activity of an
Enzyme

1. One International Unit of enzyme is defined as the amount that

catalyzes the formation of 1 micromole of product in 1 minute.

2. katal: one katal is that amount of enzyme catalyzing the conversion

of 1 mole of substrate to product in 1 second.

3. One katal = 6 X 107 International Units.

4. Specific activity:
 rectangular
hyperbola

v = Vmax[S]/(Km + [S])

Figure 13.7 Substrate saturation curve for an enzyme-catalyzed reaction. The amount of
enzyme is constant, and the velocity of the reaction is determined at various substrate
concentrations. The reaction rate, v, as a function of [S] is described by a rectangular
hyperbola. At very high [S], v = Vmax. That is, the velocity is limited only by conditions
(temperature, pH, ionic strength) and by the amount of enzyme present; v becomes
independent of [S]. Such a condition is termed zero-order kinetics. Under zero-order
conditions, velocity is directly dependent on [enzyme]. The H2O molecule provides a
rough guide to scale. The substrate is bound at the active site of the enzyme.
 13.3j Linear Plots Can Be Derived from the P450

Michaelis-Menten Equation
Be able to derive these equations
• Lineweaver-Burk:
• Begin with v = Vmax[S]/(Km + [S]) and take the
reciprocal ( 倒數 )of both sides
• Rearrange to obtain the Lineweaver-Burk equation:

(13.29)

• A plot of 1/v versus 1/[S] should yield a straight line.

Figure 13.9 The Lineweaver-Burk double-reciprocal plot.

(13.29)
 Linear Plots Can Be Derived from the Michaelis-
Menten Equation
• Hanes-Woolf:
• Begin with Lineweaver-Burk and multiply both sides
by [S] to obtain:

(13.31)

• Hanes-Woolf is best - why?

• Because Hanes-Woolf has smaller and more
consistent errors across the plot.
• Not overemphasizing the data obtained at low [S], a
fault inherent in the Lineweaver-Burk plot.
Figure 13.10 A Hanes-Woolf plot of [S]/v versus [S]

(13.31)
*Both Km and Vmax to be accurately estimated.

Computer
fitting of v
versus [S]
data to the
Michaelis-
Menten
equation is
more
commonly
done than
graphical
plotting.
 13.3k Nonlinear Lineweaver-Burk or Hanes-
Woolf Are a Property of Regulatory Enzymes
(P451)
‧Disobey the Michaelis-Menten equation
→a departure from linearity in these straight-line graphs

‧Such deviations from linearity are characteristic of the

kinetics of regulatory enzymes known as allosteric
enzymes. 異位調節酵素

‧Such regulatory enzymes are very important in the

overall control of metabolic pathways.
 13.3l Enzymatic Activity is Strongly Influenced
by pH
• Enzyme-substrate recognition and catalysis are
greatly dependent on pH.
• Enzyme— ionizable side chains and prosthetic
groups
1. determine its secondary and tertiary structure,
2. intimately involved in its active site.

• Substrate—
1. ionizing groups,
2. one or another of the ionic forms may
preferentially interact with the enzyme.
• Enzyme—
1. active over limited pH range,
2. most have a particular pH at which their catalytic
activity is optimal.
• The effects of pH may be due to effects on Km or Vmax
or both.
• Figure 13.11 Enzyme activity — pH
• The pH-activity response of an enzyme may be a
factor in the intracellular regulation of its activity.
 Figure 13.11 The pH activity profiles of four
different enzymes.

Trypsin, an intestinal protease, has slightly alkaline pH optimum, whereas

pepsin, a gastric protease, acts in the acidic confines of the stomach and has a
pH optimum near 2. Papain, a protease found in papaya, is relatively
insensitive to pHs between 4 and 8. Cholinesterase activity is pH sensitive
below pH 7 but not between pH 7 and 10. The cholinesterase pH activity
profile suggests that an ionizable group with pK' near 6 is essential to its
activity. Might it be a histidine residue within the active site?
 13.3m The Response of Enzymatic Activity to
Temperature is Complex (p453)
• Rates of enzyme-catalyzed reactions generally increase
with increasing temperature.
• However, at temperatures above 50o to 60oC, enzymes
typically show a decline in activity
• Two effects here:
• Enzyme rate typically doubles in rate for ever 10ºC as
long as the enzyme is stable and active.
• At higher temperatures, the protein becomes unstable
and denaturation occurs. Instability of higher orders of
protein structure at elevated temperature --- enzyme is
inactivated.
• Enzymes of thermophilic bacteria--- > 85oC retain full
activity Taq DNA polymerase
 Figure 13.12
The effect of temperature on enzyme activity.
The relative activity of
an enzymatic reaction
as a function of
temperature. The
decrease in the activity
above 50°C is due to
thermal denaturation.
 13.4 What Can Be Learned from the
Inhibition of Enzyme Activity?
• Enzymes may be inhibited reversibly or
irreversibly.
• Reversible inhibitors may bind at the active site or
at some other site.
• Enzymes may also be inhibited in an irreversible
manner.
• Penicillin is an irreversible suicide inhibitor.

P453
 13.4a Enzyme May Be Inhibited Reversibly
or Irreversibly
‧Enzyme inhibitors:
1. reversible inhibitors interact with the enzyme
through noncovalent association/dissociation
reactions.
2. irreversible inhibitors usually form covalent bonds
with side chains or prosthetic groups in the
enzyme.

‧The consequence of irreversible inhibition is a decrease

in the concentration of active enzyme.
 13.4b Reversible Inhibitors May Bind at the
Active Site or at Some Other Site
‧Reversible inhibitors--- two major categories:
1. competitive inhibitors: 競爭性的抑制劑
2. noncompetitive inhibitors: 非競爭性的抑制劑

‧competitive inhibitors: the substrate and inhibitor compete

for the same binding site on the enzyme.
‧active site or substrate-binding site:

‧[S] ↑ → S + enzyme instead of I + enzyme

=> High [S] can overcome the effects of I.
‧In noncompetitive inhibition => High [S] can not
overcome the effects of I.
Competitive Inhibitors Compete With Substrate for
the Same Site on the Enzyme

-1
x-intercept 
 [I] 
Km 1 + 
Figure 13.13  KI 
Lineweaver-Burk plot of competitive inhibition, showing
lines for no I, [I], and 2[I]. Note that when [S] is infinitely
large (1/[S]  0), Vmax is the same, whether I is present of
not.
P455
 琥珀酸脱氫酶
Succinate Dehydrogenase — A Classic
Example of Competitive Inhibition (P425)

P455
Figure 13.14 Structures of succinate, the substrate of succinate
dehydrogenase (SDH), and malonate, the competitive inhibitor.
Fumarate (the product of SDH) is also shown.
Noncompetitive Inhibition (P456)

→S
→ES
S
I E

↑[S] [I] ←→not affected

P456
Pure Noncompetitive Inhibition (P456)

P456
Pure Noncompetitive Inhibition – where S and I
bind to different sites on the enzyme

Figure 13.15 Lineweaver-Burk plot of pure noncompetitive

inhibition. Note that I does not alter Km but that it decreases
Vmax. In the presence of I, the y-intercept is equal to (1/Vmax)(1
+ I/KI).
Km unchanged Vmax ↓
Mixed Noncompetitive Inhibition (P456)

+I Km altered
Vmax altered
 Mixed Noncompetitive Inhibition: binding of I by E
influences binding of S by E

Figure 13.16 Lineweaver-Burk plot of mixed noncompetitive

inhibition. Note that both intercepts and the slope change in
the presence of I. (a) When KI is less than KI'; (b) when KI is
greater than KI'.
+I Km altered Vmax altered
 無競爭性的抑制劑
In Uncompetitive Inhibition, I combines only with ES

Figure 13.17 Lineweaver-

Burk plot of uncompetitive
inhibition. Note that both
intercepts change but the
slope (Km/Vmax) remains
constant in the presence
of I.

(1) Li+: ↓ depression

(2) Some pesticides
The effects of Various Types of Inhibitors on the
Michaelis-Menten Rate Equation and on Apparent
Km and Apparent Vmax
 13.4c Enzymes Also Can Be Inhibited in an
Irreversible Manner (P458)
‧If the inhibitor combines irreversibly with the enzyme
— by covalent attachment— the kinetic pattern seen is
like that of noncompetitive inhibition
— the net effects is a lost of active enzyme.
‧can be distinguished from the noncompetitive,
reversible inhibition 瞬間的 , 即時的

— the reaction of I and E (and/or ES) is not instantaneous

→ there is a time-dependent decrease in enzymatic activities
as E + I → EI proceeds, the rate of this inactivation can
be followed.
‧Unlike reversible inhibitions, dilution or dialysis of the
enzyme: inhibitor solution does not dissociate
the EI complex and restore enzyme activity.
 Suicide Substrates — Mechanism-Based Enzyme
Inactivators (P458) 自殺性受質
‧Suicide substrates are inhibitory substrate analogs
designed so that, via normal catalytic action of the
enzyme, a very reactive group is generated.
→ this reactive group then forms a covalent bond with
a nearly functional group within the active site of the
enzyme, thereby causing irreversible inhibition.
‧Suicide substrates, also called Trojan horse substrates,
are a type of affinity label. 特洛埃木馬 ; 木馬屠城計
顛覆分子
腦程式內的病毒

‧They bind with specificity and high affinity to the enzyme

active site — in their active form, they become covalently
bound to the enzyme.
‧This covalent link effectively labels a particular
functional group within the active site, identifying the
group as a key player in the enzyme’s catalytic cycle.
 Penicillin — A Suicide Substrate (P458)

‧Drugs— mechanism-based enzyme inactivators.

‧Penicillin— covalently react with an essential serine residue
in the active site of glycoprotein peptidase, an enzyme that
acts to crosslink the peptidoglycan chains during synthesis
of the bacterial cell walls (Figure 13.18). 肽聚糖

‧Once the cell wall synthesis is blocked, the bacterial cells

are very susceptible to rupture by osmotic lysis and bacterial
growth is halted.
 Penicillin — A Suicide Substrate (P458)
Figure 13.18
Penicillin is an irreversible inhibitor
of the enzyme glycoprotein
peptidase, which catalyzes an
essential step in bacterial cell wall
synthesis. Penicillin consists of a
thiazolidine ring fused to a b-lactam
ring to which a variable group R is
attached. A reactive peptide bond in
the b-lactam ring covalently attaches
to a serine residue in the active site
of the glycopeptide transpeptidase.
(The conformation of penicillin
around its reactive peptide bond
resembles the transition state of the
normal glycoprotein peptidase
substrate.) The penicilloyl-enzyme
complex is catalytically inactive. The
bond between the enzyme and
penicillin is indefinitely stable; that is,
penicillin binding is irreversible.
13.5 - What Is the Kinetic Behavior of
Enzymes Catalyzing Bimolecular Reactions?
• Enzymes often catalyze reactions involving two
(or more) substrates.
• Bisubstrate reactions may be sequential or
single-displacement reactions or double-
displacement (ping-pong) reactions.
• Single-displacement reactions can be of two
distinct classes:
逢機的1. Random, where either substrate may bind
first, followed by the other substrate
2. Ordered, where a leading substrate
有次序的
binds first, followed by the other substrate
P459
 ‧One enzyme — two (or more) substrates take
part — bisubstrate reaction

‧By one of two possible routes:

1

‧reactions of this type are defined as sequential or

single-displacement reactions.

They can be either of two distinct classes:

a. Random, where A or B may bind the enzyme first, followed
by other substrate, or
b. Ordered, where A, designated the leading substrate, must
bind to E first before B can be bound.
=> Lineweaver-Burk double-reciprocal plots (Fig.13.19)
 Figure 13.19 Single-displacement bisubstrate
mechanism.

Double-reciprocal plots of the rates observed with different fixed concentrations of one
substrate (B here) are graphed versus a series of concentrations of A. Note that, in these
Lineweaver-Burk plots for single-displacement bisubstrate mechanisms, the lines intersect to
the left of the 1/v axis.
P460
 2

‧Ping-pong or double-displacement reactions.

‧Two features:
1 the obligatory formation of a modified enzyme intermediate,
E’. 必須的

2 the pattern of parallel lines obtained in double-reciprocal

plots (see Fig. 13.22)

P430
The Double Displacement (Ping-Pong)
Reaction
Figure 13.22
Double-
displacement
(ping-pong)
bisubstrate
mechanisms are
characterized by
parallel lines.
 13.5a The Conversion of AEB to PEQ is the Rate-
Limiting Step in Random, Single-Displacement
Reactions
E rapidly
A, B, P, Q AE, EB, PE, EQ
Reversibly

(13.49)

In this type of sequential reaction, all possible binary enzyme-

substrate and enzyme-product complexes are formed rapidly and
reversibly when enzyme is added to a reaction mixture
containing A, B, P, and Q.
• If A has no influence on the binding constant for B (and vice
versa); the mechanism is purely random, then the lines in a
Lineweaver-Burk plot intersect at the 1/[A] axis (Fig. 13.20).

Figure 13.20
Random, single-displacement bisubstrate mechanisms where A does not affect
B binding, and vice versa. Note that the lines intersect at the 1/[A] axis. (If [B] were
varied in an experiment with several fixed concentrations of A, the lines would
intersect at the 1/[B] axis in a 1/v versus 1/[B] plot.)
 Creatine Kinase Acts by a Random, Single-
Displacement Mechanism
‧An example, creatine kinase, a phosphoryltransfer enzyme
that uses ATP as a phosphoryl donor to form creatine
phosphate (CrP) from creatine (Cr).
‧Creatine-P is an important reservoir of phosphate-bond energy
in muscle cells (Figure 13.21).

The overall direction of the reaction will be determined by

the relative concentrations of ATP, ADP, Cr, and CrP and
the equilibrium constant for the reaction.
‧Direction of the reaction — by [ATP], [Cr], [CrP]& k .
P461
‧Enzymes has two sites for substrate (or product) binding:
(1) An adenine nucleotide site, where ATP or ADP binds,
(2) A creatine site, where Cr or CrP is bound.
compete
compete

‧No E-PO4 intermediate appears.

‧rapid & reversibly binary ES complex formation
→ the addition of the remaining substrate
→ the rate-determining reaction take place within the ternary
complex.
Figure 13.21 The structures of creatine and
creatine phosphate, guanidinium compounds that
are important in
muscle energy
metabolism.
 13.5b In an Ordered, Single-Displacement
Reaction, the Leading Substrate Must Bind First

‧The leading substrate, A (also called the obligatory or

compulsory substrate), must bind first, then the second
substrate, B.
‧One representation, suggested by W.W. Cleland, follows:

The leading substrate (A) binds first, followed by B. (13.50)

Reaction between A and B occurs in the ternary complex
and is usually followed by an ordered release of the
products, P and Q.
 An Alternative way of Portraying the Ordered,
Single-Displacement Reaction

‧A & P are competitive for binding to the free enzyme, E,

but not A and B (or P and B).
 NAD+-Dependent Dehydrogenases Show Ordered
Single-Displacement Mechanisms
‧NAD+-dependent dehydrogenases are enzymes

‧The leading substrate (A) is NAD+.

‧NAD+ & NADH (product P) compete for a common site on E.

‧Example, alcohol dehydrogenase (ADH):

‧No B (ethanol) is bound to E is the absence of A
(NAD+)
→ ordered mechanism → Not random.
13.5c The Double Displacement (Ping-Pong)
Reactions Proceed Via Formation of a Covalently
Modified Enzyme Intermediate

Double-Displacement (Ping-Pong) reactions proceed via

formation of a covalently modified enzyme intermediate (E’).
Reactions conforming to this kinetic pattern are characterized
by the fact that the product of the enzyme’s reaction with A
(called P in the above scheme) is released prior to reaction of
the enzyme with the second substrate, B.
 An Alternative Presentation of the Double-
Displacement (Ping-Pong) Reaction

→ A & Q compete for free enzyme form E. while

B & P compete for the modified enzyme form, E’ the enzyme
→ A & Q do not bind to E’, nor do B and P combine with E.
 The Double Displacement (Ping-Pong)
Reaction
Figure 13.22
Double-
displacement
(ping-pong)
bisubstrate
mechanisms are
characterized by
parallel lines.
 Aminotransferases Show Double-Displacement
Mechanisms
‧One class of enzymes — ping-pong type mechanism —
aminotransferases

Amino acid -keto acid

Amino group
Amino group
Amino acid 1 + keto acid 2 → keto acid 1 + amino acid 2
‧A specific example would be glutamate: aspartate
aminotransferase.

‧Figure 13.23 mechanism

 Glutamate:aspartate Aminotransferase
Figure 13.23
Glutamate:aspartate
aminotransferase, an
enzyme conforming to
a double-displacement
bisubstrate
mechanism.
Glutamate:aspartate
aminotransferase is a
pyridoxal phosphate-
dependent enzyme.
The pyridoxal serves
as the -NH2 acceptor
from glutamate to form
pyridoxamine.
Pyridoxamine is then
the amino donor to
oxaloacetate to form
asparate and
regenerate the
pyridoxal coenzyme
form. (The
pyridoxamine: enzyme
is the E' form.)
‧Glutamate and aspartate are competitive for E &
oxaloacetate & -ketoglutarate compete for E’

‧Not all enzymes displaying ping-pong-type mechanisms

require coenzymes as carriers for the chemical substituent
transferred in the reaction. 取代者 ; 【化】置換基 ; 取代基
 13.5d Exchanged Reactions Are One Way to
Diagnose Bisubstrate Mechanisms (P464)
‧Assign a reaction mechanism to a specific enzyme.
‧exchange reaction (see text for detail)
交換反應
 13.5e Multisubstrate Reactions Can Also Occur in
Cells
‧More than two substrates?

‧An example, the glycolytic enzyme

glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
NAD+ + glyceraldehyde-3-P + Pi

NADH + H+ + 1, 3-bisphosphoglycerate
大量存在
‧Many other multisubstrate examples abound in metabolism.

This is the end of 13-2.

 補充教材
Viagra – An Unexpected Outcome in a Program of
Drug Design

Note the similarities in the structures of cGMP (left) and

Viagra (right). Viagra produces an increase in [cGMP] in
penile vascular tissue, allowing vascular muscle relaxation,
improved blood flow, and erection.