CHAPTER

6
Enzymes

Key topics about enzyme function:

Physiological significance of enzymes
Chemical mechanisms of catalysis
Mechanisms of chymotrypsin
Description of enzyme kinetics and inhibition
 What are enzymes?
Enzymes are catalysts
Increase reaction rates without being used up
Most enzymes are globular proteins
However, some RNA (ribozymes and ribosomal RNA) also
catalyze reactions
Study of enzymatic processes is the oldest field of
biochemistry, dating back to late 1700s
The Buchner demonstrated for the first time that
fermentation: could take place outside living cells (vital
force).
enzyme (Greek: en, in + zyme, yeast)
Study of enzymes has dominated biochemistry in the
past and continues to do so
 Proteins are:
Polypeptides (covalently linked a-amino acids) + possibly:
cofactors
- functional non-amino acid component
- metal ions or organic molecules
coenzymes (derived from vitamins, often donates or receives
electrons or functional group during the reaction)
- organic cofactors
- NAD+ in lactate dehydrogenase
prosthetic groups (A coenzyme or metal ion that is very tightly
or even covalently bound to the enzyme protein)
- heme in myoglobin

A complete, catalytically active enzyme together with its bound

coenzyme and/or metal ions is called a holoenzyme.
The protein part of such an enzyme is called the apoenzyme or apoprotein.
 Pearson Education
Ltd 2015. Copying
permitted for
Examples of chemical and enzymatic cleavage of the
peptide bond

ChemicalChemical
reactionReaction

bond cleaved

O O 6 M HCl O
110 o C + +
+
NH3 CH C N
H
CH C OH NH3 CH C OH + NH3 CH COOH
6 hr
CH2 CH3 CH2 CH3

O O

OH OH

Enzymatic Reaction
Enzymatic reaction
bond cleaved

O O chymotrypsin
pH 7.5
COO -
+
COO - +
+
O-
+ C CH NH3 CH
NH3 CH N CH C NH3
o
H 37 C
CH2 CH3 ~ min CH2 CH3

O O

OH OH 8
Reaction Coordinate Diagram

Enzymatic Reaction

bond cleaved

O O chymotrypsin
pH 7.5
COO -
+
COO - +
+
O-
+ C CH NH3 CH
NH3 CH N CH C NH3
o
H 37 C
CH2 CH3 ~ min CH2 CH3

O O

OH OH
 D
How to Lower G

Enzymes bind transition states best

The idea was proposed by Linus Pauling in 1946
Enzyme active sites are complimentary to the
transition state of the reaction
Enzymes bind transition states better than substrates
Stronger/additional interactions with the transition
state as compared to the ground state lower the
activation barrier
 An enzyme with a pocket complementary to the reaction
transition state helps to destabilize the stick, contributing to
catalysis of the reaction.
Many enzymes undergo a conformational change when the
substrates are bound to the active site; this change is called
an induced fit.
 Catalytic Mechanisms
make the reaction go faster

acid-base catalysis: give and take protons

covalent catalysis: moving electrons
Electrostatic catalysis: charged molecules and metal ions
Proximity and orientation effects: make collisions
Enzymatic Reaction

bond cleaved

O O chymotrypsin
pH 7.5
COO -
+
COO - +
+
O-
+ C CH NH3 CH
NH3 CH N CH C NH3
o
H 37 C
CH2 CH3 ~ min CH2 CH3

O O

OH OH
Active Site of Chymotrypsin

14
Catalytic Mechanism- Serine Proteases

15
16
Serine proteases catalyze the hydrolysis of the
peptide bond
Serine Protease Family:
Enzymes that utilize the same catalytic mechanisms are generally grouped into families
The proteases:

His 57
Chymotrypsin
Ser 195
Trypsin
Elastase
O CH2O
Asp 102
CH2C HN N
Serine Proteases have a Charge Transfer Relay Network: H. O
O
-

Consisting of an aspartic acid, histidine, and serine residues

These residues participate directly in the cleavage of the peptide bond
Charge transfer relay network for chymotrypsin

17
Specificity of Serine Proteinases

18
specificity pockets

19
 References for enzyme kinetics
Elementary kinetics
http://www.wiley.com/legacy/college/boyer/0470003790/reviews/kinetics/kinetics_intro.htm

Enzyme catalysis:
http://www.wiley.com/legacy/college/boyer/0470003790/animations/catalysis_energy/catalysis_energy.htm

Enzyme kinetics
http://www.chem.purdue.edu/courses/chm333/enzyme_kinetics.swf

Enzyme inhibitors:
http://www.chem.purdue.edu/courses/chm333/enzyme_inhibition.swf

Enzyme inhibitors_2
http://www.wiley.com/legacy/college/boyer/0470003790/animations/enzyme_inhibition/enzyme_inhibition.htm
The study of enzyme catalysis has become important
for drug development

The study of the rates of enzyme-catalyzed reactions is

called enzyme kinetics

kinetic description of enzyme activity will help us

understand how enzymes function
Elucidate acid-base catalysis
Understand catalytic mechanism

Many drugs work by inhibiting an enzyme.

 Enzymes
Vocabulary

Chemistry Biochemistry
Reactant Substrate (S)
Catalyst Enzyme (E)
Enzyme and substrate combine to form the enzyme-substrate
complex, this complex yields the product and degrades to
regenerate free enzyme.

S
Enzyme
Enzyme and substrate combine to form the enzyme-
substrate complex, this complex yields the product
and degrades to regenerate free enzyme.

Enzyme
Enzyme and substrate combine to form the enzyme-substrate
complex, this complex yields the product and degrades to
regenerate free enzyme.

Enzyme
 What is the evidence for the existence of an
enzymesubstrate complex?
At a constant concentration of enzyme, the reaction rate increases with
increasing substrate concentration until a maximal velocity is reached

Zero-order

First-order

What reaction mechanism might explain these data?

What do we mean when we say the
rate of a chemical reaction?
Rate a [reactants]

Rate = k [reactants]

where k is a proportionality constant, the rate constant.

Rate = kf [reactants] = kf [A] [B]

Initial velocity for the reaction V0 = kf [A] [B]

V0 = rate = (total product formed) / (time elapsed)

MichaelisMenten equation

Rate of ES formation = Rate of ES breakdown

 Does the equation fit experimental observations?

Yes; we can confirm this by considering the limiting situations where [S] is
very high or very low
An important numerical relationship emerges from the Michaelis-
Menten equation in the special case when
V0 is exactly one-half Vmax
 The constant Km has two meanings

2.
Km = [S] when

When k2 is rate-limiting, k2 << k-1 and Km reduces

to k-1/k1, which is defined as the dissociation
constant, Kd, of the ES complex
 The maximal rate, Vmax, reveals the turnover
number of an enzyme
which is the number of substrate molecules converted into product by an
enzyme molecule in a unit time when the enzyme is fully saturated with
substrate

If an enzyme reacts by the two-step Michaelis-Menten mechanism,

Vmax = k2[Et] kcat =Vmax/[Et] the turnover number.

How are Km and Vmax determined experimentally?

-------------------------
The Michaelis-Menten equation can be algebraically transformed
into versions that are useful in the practical determination
of Km and Vmax
 The Problem:

The velocity of an enzyme-substrate reaction was

measured at several substrate concentrations.

[S] (M) (mMs-1)

0.25 0.75
0.5 1.20
1.0 1.71
2.0 2.18
4.0 2.53

Calculate KM and Vmax for the reaction?

34
 The solution:

Calculate the reciprocals of the substrate

concentration and velocity as in table
below.
-1
1/[S] (M ) 1/ (mM .s) -1

4.0 1.33
2.0 0.83
1.0 0.58
0.5 0.46
0.25 0.40

35
Plot a Lineweaver-Burk graph, that is, 1/ versus
1/[S] as shown below.

Intercept on the 1/[S] axis (which is equal to -1/KM ) is -1.33 M-1

1
KM = -( ) = 0.75 M
- 1.33 M -1
Intercept on the 1/ axis (which is equal to 1/ Vmax)
is 0.33 mM -1.s. Therefore,

1
V max = -1
0.33 mM .s
= 3.0 mM.s
-1
Enzyme inhibition can be classed into four types on the basis
of slope and intercept effects

1. Competitive
Do not form covalent bonds with
2. Uncompetitive
the enzyme
3. Mixed Inhibition or Noncompetitive
4. Irreversible Inhibition Inhibitor covalently binds enzyme

Reversible inhibitors bind to and can dissociate from the enzyme

They are often structural analogs of substrates or products
They are often used as drugs to slow down a specific enzyme

Reversible inhibitor can bind:

to the free enzyme and prevent the binding of the substrate
to the enzyme-substrate complex and prevent the reaction

Irreversible inhibitors (inactivators) react with the enzyme

One inhibitor molecule can permanently shut off one enzyme molecule
They are often powerful toxins but also may be used as drugs
 Competitive inhibitors

Page 379
In LineweaverBurk plot y-intercept represents the rate of an enzymatic reaction at
very high concentrations of substrate, whereas the slope represents the rate at very
low concentrations of substrate.
 Competitive inhibitors

affect the slope of a LineweaverBurk plot but do not alter the y-intercept
Therefore, a competitive inhibitor only binds to the enzyme at very low
concentrations of substrate (i.e., an effect on Vmax/Km, which is the
reciprocal of the slope).
However, at infinitely high levels of substrate the inhibitor does not bind
(i.e., no effect on Vmax, which is the reciprocal of the y-intercept).
 uncompetitive inhibitors

In contrast to competitive, uncompetitive inhibitors only affect the y-intercept of a

LineweaverBurk plot and do not alter the slope
Therefore, an uncompetitive inhibitor only binds to the enzyme at infinitely high
substrate concentration (i.e., an effect on Vmax, the reciprocal of the y-intercept)
but at very low concentrations of substrate the inhibitor does not bind (i.e., no
effect on the slope).
This agrees exactly with the mechanistic scheme for uncompetitive inhibition
where the inhibitor only binds to the ES complex.
 noncompetitive inhibitor

binds to both the free enzyme (E) and the ES complex, it will affect the y-
intercept of a LineweaverBurk plot
Therefore, a noncompetitive inhibitor binds to an enzyme when the
varied substrate is either at very low or very high concentrations.
In essence it is a combination of competitive and uncompetitive
inhibition.

[I]
 Chapter 6: Summary
In this chapter, we learned:

why nature needs enzyme catalysis

how enzymes can accelerate chemical reactions
how chymotrypsin breaks down peptide bonds
how to perform and analyze kinetic studies
how to characterize enzyme inhibitors
how enzyme activity can be regulated