6| Enzymes

© 2017 W. H. Freeman and Company

 CHAPTER 6
Enzymes

Learning goals:
• Physiological significance of enzymes
• Origin of catalytic power of enzymes
• 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
• Study of enzymes has dominated biochemistry in the
past and continues to do so
Enzyme-Substrate Complex
Definition:
A protein or other molecule that acts as a catalyst for a
biological reaction

Enzymes work by lowering Activation Energy of reaction

therefore : Speeds up the reaction

Active Site: a pocket in an enzyme with the specific shape

and chemical make up necessary to bind a substrate
Many enzymes are specific to a substrate or reaction eg:
catalase (very fast & very specific)

Enzyem Co-Factors:
Non-protein part of an enzyme essential to catalytic
activity

Eg: Fe, Cu, Mn, Mo, Co, Ni, Se, Zn, Ca (minerals)

Often held by side chains of amino acids

Co-Enzyme :

An organic molecule that acts as an enzyme co-factor

Eg: Water Soluble Vitamins: Niacin (B3), Riboflavin

(B2), Ascorbic acid (C), B6, B12

Oil Soluble vitamins are NOT Co-Enzyme

1) Oxido- Reductases

a) dehaydrogenases:

• removal of H from substrate to another

molecule but not O2
useful in electron transport
AH2 + B A + BH2

b) Oxidases

• At the end of oxidation

AH2 + ½ O2 A + H2O
2) Transferases

Transfer of chemical groups

( NH2,OH,PO4) from one substrate to
another.

Eg: transaminase
3) Hydrolases

Split bonds and add H2O

Eg: proteases, trypsin, lipase, phosphatases,
amylase, nucleases
4)Lyases

Nonhydrolytic breaking of various

chemical bonds

Often forming a new double bond or a

new ring structure

C=C C=O C=N

Eg: decaroboxylases
5) Ligases

Use ATP to form bonds

eg: Phosphorylation
repair of DNA – after cutting

Glocose + ATP glucose -6-phosphate

(same bonds, different location)

6) Isomerases

Rearrangement of molecules

Glucose-6-phosphate fructose-6-phosphate

Dihydroxyaceton glyceraldehyde
 Enzymatic Catalysis
• Enzymes do not affect equilibrium (Keq)…
• …which means that enzymes cannot affect the
free energy of the reaction (ΔG).
• Slow reactions face significant activation
barriers (ΔG‡) that must be spending during
the reaction.
• Enzymes increase reaction rates by
decreasing ΔG‡.
Reaction Coordinate Diagram
Enzymes Decrease ΔG‡
 Rate Enhancement by Enzymes
Some Rate Enhancements Produced by
TABLE 6-5
Enzymes
Cyclophilin 105
Carbonic anhydrase 107
Triose phosphate isomerase 109
Carboxypeptidase A 1011
Phosphoglucomutase 1012
Succinyl-CoA transferase 1013
Urease 1014
Orotidine monophosphate decarboxylase 1017
Illustration of TS Stabilization Idea:
Imaginary Stickase
 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
 Role of binding energy in catalysis. To lower
the activation energy for a reaction, the system
must acquire an amount of energy equivalent to
the amount by which ΔG‡ is lowered. Much of
this energy comes from binding energy (ΔGB)
contributed by formation of weak noncovalent
interactions between substrate and enzyme in the
transition state.
 What is enzyme kinetics?
• Kinetics is the study of the rate at which compounds
react
• Rate of enzymatic reaction is affected by:
– enzyme
– substrate
– effectors
– temperature
 Why Study Enzyme Kinetics?

• Quantitative description of biocatalysis

• Determine the order of binding of substrates
• Elucidate acid-base catalysis
• Understand catalytic mechanism
• Find effective inhibitors
• Understand regulation of activity
 Carry Out the Algebra
• The final form in case of a single substrate is the Michaelis-Menten equation:

k cat [E tot ][S] V max[S]

v 
K m  [S] Km  [S]
• kcat (turnover number): how many substrate molecules one enzyme molecule can
convert per second
• Km (Michaelis constant): an approximate measure of a substrate’s affinity for an
enzyme
• During steady state, the maximum velocity (Vmax) occurs when all of the enzyme
is in the ES complex and is dependent on the breakdown of that complex
(k[ES]).
• The microscopic meaning of Km and kcat depends on the details of the
mechanism.
 Saturation Kinetics:
At high [S] velocity does not depend on [S]
Determination of Kinetic Parameters

Nonlinear Michaelis-Menten plot should be used to

calculate parameters Km and Vmax.

Linearized double-reciprocal plot is good for analysis

of two-substrate data or inhibition.
 Lineweaver-Burk Plot:
Linearized, Double-Reciprocal
 Enzyme efficiency is limited by diffusion:
kcat/KM

• Can gain efficiency by having high velocity or affinity for substrate

– Catalase vs. acetylcholinesterase
 Enzyme Inhibition
Inhibitors are compounds that decrease enzyme’s activity

• 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

• 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
1) Irreversible Inhibition

Usually covalent bond permanently attached

to OH or SH in active site

e.g Heavy metals (Ag+,Hg+2, Cu+2 ) not good

usually
react with NH2 and COOH too

eg. Cyanide – CN
eg. Nerve Gases/Insecticides
Nerve gases
-all work in similar (deadly) ways
BUT : how do they ‘work;?
When a normally functioning motor nerve is stimulated, it
releases the acetylcholine, which transmits the impulse to
a muscle or organ. Once the impulse is sent, the enzyme
acetylcholinesterase immediately breaks down the
acetylcholine in order to allow the muscle or organ to
relax.

Nerve agents attack the nervous system of the human

body.
All such agents function the same way: by inhibiting the
enzymeacetylcholinesterase, which is responsible for the
breakdown of acetylcholine (ACh) in the synapse.. ACh
gives the signal for muscles to contract, preventing them
from relaxing.
 Diisopropylfluorophosphate
(DIFP)

Irreversible inhibition.
Reaction of chymotrypsin with
diisopropylfluorophosphate (DIFP), which
modifies Ser195, irreversibly inhibits the
enzyme. This has led to the conclusion that
Ser195 is the key active-site aa residue in
chymotrypsin.
2) Reversible Inhibition – you may survive

Three Types: competitive, uncompetitive,

Mixed inhibition
 Competitive Inhibition
• Competes with substrate for binding
– binds active site
– does not affect catalysis

• No change in Vmax; apparent increase in KM

• Lineweaver-Burk: lines intersect at the y-axis.
Competitive Inhibition
 Uncompetitive Inhibition
• Only binds to ES complex
• does not affect substrate binding
• inhibits catalytic function

• Decrease in Vmax; apparent decrease in KM

• No change in KM/Vmax
• Lineweaver-Burk: lines are parallel.
Uncompetitive Inhibition
 Mixed Inhibition

• Binds enzyme with or without substrate

― binds to regulatory site
― inhibits both substrate binding and catalysis
• Decrease in Vmax; apparent change in KM
• Lineweaver-Burk: lines intersect left from the y-axis.
• Noncompetitive inhibitors are mixed inhibitors such that
there is no change in KM.
Mixed Inhibition
(The pH effect on Enzyme)
The pH-activity profiles of two enzymes.. Because pH is a
logarithmic scale reflecting tenfold changes in [H+], the
changes in V0 are also plotted on a logarithmic scale.
The pH optimum for the activity of an enzyme is generally
close to the pH of the environment in which the enzyme is
normally found.
Pepsin, which hydrolyzes certain peptide bonds of proteins
during digestion in the stomach, has a pH optimum of about
1.6. The pH of gastric juice is between 1 and 2.
Glucose 6-phosphatase of hepatocytes (liver cells), with a
pH optimum of about 7.8, is responsible for releasing
glucose into the blood. The normal pH of the cytosol of
hepatocytes is about 7.2.
The pH dependence of chymotrypsin-catalyzed reactions.
(a) The rates of chymotrypsin-mediated cleavage produce a bell-
shaped pH-rate profile with an optimum at pH 8.0.
The rate (v) being plotted is that at low substrate concentrations and
thus reflects the term kcat/Km. The plot can be broken down to its
components by using kinetic methods to determine the terms kcat and
Km separately at each pH. When this is done (b and c), it becomes clear
that the transition just above pH 7 is due to changes in kcat, whereas the
transition above pH 8.5 is due to changes in 1/Km.
Kinetic and structural studies have shown that the transitions illustrated
in (b) and (c) reflect the ionization states of the His57 side chain (when
substrate is not bound) and the α-amino group of Ile16 (at the amino
terminus of the B chain), respectively. For optimal activity, His57 must
be unprotonated and Ile16 must be protonated.