ENZYMES

Enzymes (History)

• First discovered by Eduard Buchner in 1897

who observed that yeast extracts can ferment
sugar to alcohol
• This proved that fermentation was promoted
by molecules that continued to function when
removed from cells
• The first enzyme to be purified and
crystallized was urease in 1926; these
crystals consisted entirely of protein
• Later, pepsin, trypsin and other digestive
proteins were isolated and determined to be
purely protein as well
 Enzymes

 Enzymes are the catalysts of nature.

 With the exception of catalytic RNA, all enzymes are

proteins.

 Catalyst alter the rate of a chemical reaction without

undergoing a permanent change in structure.

 Catalytic activity is dependant upon native

conformation; if it is lost, then catalytic activity is
lost as well

 All levels of protein architecture must be intact and

correct for enzymes to perform their functions

 They range in molecular weights from 12,000 to over

1 million
Enzyme Classification

Simple Enzymes: composed of whole proteins

Complex Enzymes: composed of protein plus a relatively small

organic molecule

holoenzyme = apoenzyme + prosthetic group / coenzyme

A prosthetic group describes a small organic or metalloorganic molecule

bound to the apoenzyme by covalent bonds.

When the binding between the apoenzyme and non-protein

components is non-covalent, the small organic molecule is called a
coenzyme.

Coenzymes serve as transient carriers of specific functional groups.

They often come from vitamins (organic nutrients required in small amounts in the
diet)
How enzymes work
• Enzymes provide specific environments in which
chemical reactions that don’t normally proceed under
neutral pH, mild temperature, and aqueous
environment conditions can occur
• This region is a pocket on the enzyme known as the
active site
• The molecule that is bound to the active site and
acted upon by the enzyme is called the substrate
• The two together form what is known as the enzyme-
substrate complex
• The function of an enzyme catalyst is to increase the
rate of a chemical reaction, not affect is equilibrium
• Therefore, enzymes don’t make more product, they
just make product faster
 Active Site
• The area of an enzyme that binds to the substrate
• Structure has a unique geometric shape that is designed to
fit the molecular shape of the substrate
• Each enzyme is substrate specific
• Thus the active site that is complementary to the geometric
shape of a substrate molecule

• Active site is lined with residues and sometimes contains a co-

factor
• Active site residues have several important properties:
– Charge [partial, dipoles, helix dipole]
– pKa
– Hydrophobicity
– Flexibility
– Reactivity
 Substrate Binding specificity

Complementarity
• Geometric
• Electronic (electrostatic)
• Stereospecificity (enzymes and
substrates are chiral)

1. Lock and Key model

2. Induced Fit model
 Enzyme active site

•Chymotrypsin (Cuts next to Hydrophobic Groups)

•Trypsin (Cuts next to Arg & Lys)
•Elastase (Cuts next to Val & Thr)
 Lock and Key Model

• An enzyme binds a substrate in a region called the active site

• Only certain substrates can fit the active site

• Amino acid R groups in the active site help substrate bind

Induced Fit Model

• Enzyme structure flexible, not rigid

• Enzyme and active site adjust shape to bind substrate

• Increases range of substrate specificity

• Shape changes also improve catalysis during reaction

- transition-state like configuration
Enzyme-Substrate Interaction
 hydrolysis
Sucrose Glucose and Fructose
sucrase
The function of an enzyme catalyst is to
increase the rate of a chemical reaction, not
affect its equilibrium.

Therefore, enzymes don ’ t make more

product, they just make product faster.
DG# for uncatalyzed reaction = 107 kJ
DG# for catalyzed reaction = 47 kJ
kuncat = e-107000/8.314x298
DG = -RT ln K
#/RT
kcat = e-47000/8.314x298
k = e-DG
 How can an enzyme reduce the activation energy?

(1) Binding to the substrate can be done such that the formation
of the transition state is favored
(2) Orientation and positioning of substrate(s)
(3) Bonds in the substrate can be ‘activated’ by functional groups
in the catalytic site
 Enzyme Kinetics

¾¾ ®
k1
E + S ¬¾¾ ES ¾¾® E + P
k2
k -1

E = Enzyme S = Substrate P = Product

ES = Enzyme-Substrate complex
k1 rate constant for the forward reaction
k-1 = rate constant for the breakdown of the ES to
substrate
k2 = rate constant for the formation of the products
 ¾¾ ®
k1
E + S ¬¾¾ ES ¾¾® E + P
k2
k -1
When the substrate concentration becomes large enough to
force the equilibrium to form completely all ES the second
step in the reaction becomes rate limiting because no more ES
can be made and the enzyme-substrate complex is at its
maximum value.

d P
 k 2 ES
[ES] is the difference between the ES
v formation and its disappearance.
dt

d ES
1

 k1 E S  k 1 ES  k 2 ES

dt
 Assumption of equilibrium
k-1>>k2 the formation of product is so
much slower than the formation of the ES
complex. That we can assume:

k 1 E S
Ks  
k1 ES
Ks is the dissociation constant for the ES complex.
 Assumption of steady state
Transient phase where in the course of a reaction the
concentration of ES does not change

d ES 
0
dt
 ET  E  ES 3

Combining 1 + 2 + 3

k1 ET - ESS  k -1  k 2 ES

rearranging

ESk-1  k 2  k1S  k1ET S

Divide by k1 and solve for [ES] Where
k -1  k 2
ES  E T S KM 
k1
K M  S
  d P  k2 ET S
vo     k2 ES 
 dt t 0 K M  S

vo is the initial velocity when the reaction is just starting out.

And Vmax  k 2 ET is the maximum velocity

Vmax S The Michaelis - Menten

vo 
K M  S
equation
 Michaelis – Menten Kinetics

low [S], v is proportional to [S] - first order

high [S], v is independent of [S] - zero order

The Km is the substrate concentration where vo equals one-half Vmax

 The KM widely varies among different
enzymes

The KM
k 1 k 2 k2
can be expressed as: KM    Ks 
k1 k1 k1

As Ks decreases, the affinity for the substrate

increases. The KM can be a measure for substrate
affinity if k2<k-1
 The double reciprocal plot
- in order to change this equation to a form we can use in
our analysis of enzymatic rate constants, we invert both
sides of the equation:

V0 = Vmax [S] 1 = Km + [S]

Km + [S] V0 Vmax [S]

Lineweaver-Burk Plot

1 = Km 1 1
1/V0

+
V0 Vmax [S] Vmax
Slope = Km/Vmax
-1/Km

1/Vmax
0 1/[S]
 Lineweaver-Burk plot: slope = KM/Vmax,
1/vo intercept is equal to 1/Vmax
the extrapolated x intercept is equal to -1/KM
For small errors in at low [S] leads to large errors in 1/vo

kcat is how many reactions an

Vmax enzyme can catalyze per second
kcat 
ET The turnover number
Km High Km means strength of binding is low
Relates to how strongly an enzyme binds its substrate

kcat High kcat means high speed of catalysis

Relates to how rapid a catalyst the enzyme is

Vmax High Vmax means high rate of catalysis

Related to kcat and [E] by: Vmax=kcat[E]
• kcat = turnover number; kcat = Vmax/[E]T

• kcat/Km is a measure of activity, catalytic efficiency

KM is a useful indicator of the affinity of an enzyme

for the substrate

A low KM indicates a high affinity for the substrate

A high kcat/KM ratio implies an efficient enzyme

This could result from: Large kcat

Small KM
 Enzyme Inhibition
• Inhibitors: compounds that decrease activity of the enzyme
• Can decrease binding of substrate (affect KM), or turnover #
(affect kcat) or both
• Most drugs are enzyme inhibitors
• Inhibitors are also important for determining enzyme
mechanisms and the nature of the active site.
• Important to know how inhibitors work – facilitates drug design,
inhibitor design.

• Antibiotics inhibit enzymes by affecting bacterial

metabolism
• Nerve Gases cause irreversible enzyme inhibition
• Insecticides – choline esterase inhibitors
• Many heavy metal poisons work by irreversibly
inhibiting enzymes, especially cysteine residues
 Types of Enzyme Inhibition

• Reversible inhibition
reversibly bind and dissociate from enzyme,
activity of enzyme recovered on removal of
inhibitor - usually non-covalent in nature
– Competitive
– Uncompetitive
– Noncompetitive (Mixed)

• Irreversible inhibition
inactivators that irreversibly associate with
enzyme
activity of enzyme not recovered on removal -
usually covalent in nature
 Competitive Inhibition

Inhibitor competes for the substrate binding site –

most look like substrate
substrate mimic / substrate analogue
Competitive Inhibition
Competitive Inhibition
 Uncompetitive Inhibition

Uncompetitive inhibitors bind at a site distinct from the substrate

active site and bind only to the ES complex
 Uncompetitive Inhibition

• Active site distorted after binding of S ( usually occurs

in multisubstrate enzymes) Decreases both KM and kcat
• Vo = Vmax[S]/(KM + ’[S]) K’I = [ES][I]/[ESI]
• Cannot be reversed by increasing [S] – available
enzyme decreases
 Mixed (Noncompetitive) Inhibition
• Inhibitor can bind at a site distinct from the substrate
active site to either E or ES
 Mixed Inhibition

• Vo = Vmax[S]/(KM + ’[S])

• Vmax decreases; KM can go up or down.