**ENZYMES**

Definitions--

Chemical reactions in cells require

specific catalysis.
Enzymes are proteins which perform
this function.
Metabolite acted upon is called the
enzyme’s substrate.
 A. Fundamental Properties
1) Enzymes are excellent catalysts,
speeding up reactions 108 to 1020 fold.
They speed up reactions without being
used up.
2) Specificity
a) for substrate - ranges from absolute
(e.g., aspartase) to relative
b) for reaction catalyzed, i.e.,few
side-reactions and by-products, etc.)
3) Regulated-- some enzymes can sense
metabolic signals.
 B. Enzymes as Molecules
1) Large molecules-- proteins from
12kDa - 1,000kDa or more -- most are
much larger than their substrate.

2) Active site -- specific region

in enzyme which interacts
with its substrate.
both binding and catalytic
reaction occur here.
some residues involved in binding substrate
others catalyze reaction
3) Cofactors

for some reactions, the amino acids are

not powerful enough for catalysis.
some enzymes incorporate additional
factors.
metal ions as cofactors-- Zn2+, Fe2+,
Cu2+, others
coenzymes are organic cofactors
prosthetic groups are covalently attached
C. Classification of Enzymes

1) named and classified according to the

substrate acted upon and the reaction
catalyzed.
2) trivial names-- end in -ase -- urease,
hexokinase.
3) named based on a formal systemic
catalog (IUB) with six major classifications.
(All enzymes should fall into one of these
categories and all enzymes therefore have
a formal name.)
Class 1. Oxidoreductases- catalyze redox
processes

Example: RCH2-OH  RCH=O

Class 2. Transferases- transfer chemical groups

from one molecule to another or to another part
of the same molecule.
O O
Example: CH3-C-SCoA + XR  CH3-C-XR
+ HSCoA
acetyl CoA acetyl group transferred
Class 3. Hydrolases- cleave a bond using water
to produce two molecules from one.

O H2O O
example: --CNH-R  --C-OH + H2N-R
cleavage of a peptide bond

Class 4. Lyases- remove a group from or add a

group to double bonds.

H-X H X
---C=C---  ---C--C---
Class 5. Isomerases- interconvert isomeric
structures by molecular rearrangements.
CH3 CH3
HC-OH HO-CH
COOH COOH
Class 6. Ligases -- join two separate molecules
by the formation of a new chemical bond usually
with energy supplied by the cleavage of an ATP.
example:
O ATP ADP+Pi O
-OOC-C-CH + CO -OOC-C-CH -COO-
3 2 2
pyruvate oxaloacetate
enzyme = pyruvate carboxylase
 Enzyme Mechanism

Enzymes catalyze difficult reactions by

changing the reaction to a series of “easier”
steps including
nucleophilic attack, general acid-base
catalysis, covalent attachment, etc.

Most common detailed example is action

of chymotrypsin, a protease.
 Coenzymes and Vitamins
Some coenzymes are loosely held or
transiently bound acting more or less as
second substrates & others are tightly
held within the protein. The latter are called
prosthetic groups.

Water-Soluble Vitamins
 Coenzyme = thiamine pyrophosphate (TPP)

 use = decarboxylation
and transketolation
its vitamin =
thiamine or
vitamin B1,
contains pyrimidine
and thiazole.
disease = beri-beri, Wernike’s disease.
peripheral nerves, muscle cramps, numbness
 riboflavin
Coenzyme =
flavin mononucleotide
(FMN),
flavin adenine
dinucleotide (FAD)
both act as prosthetic groups
-- use = redox reactions
-- its vitamin = riboflavin or B2
nicotinamide-adenine dinucleotide (NAD),
nicotinamide-adenine dinucleotide phosphate
(NADP) Coenzyme
-- use = redox
reactions with
H transfer
-- its vitamin = P
niacin or B3 =
nicotinamide
& nicotinic acid
-- disease =
pellagra, skin lesions,
swollen tongue, nervous/mental disorders
Coenzyme = pyrodoxal phosphate
-- use = decarboxylations, transaminations
and racemases
-- its vitamin = pyridoxine, or vitamin B6
Coenzyme = Coenzyme A (CoA)
-- use = activates carbonyl groups
and in acyl transfer
(acetyl- CoA, synthesis
of fats and steroids)
-- its vitamin = pantothenic acid -- disease= GI
problems, emotional instability, burning sensation
in extemities

acetyl
Acetyl CoA
Coenzyme = folate or tetrahydrofolate (the
reduced form)
-- use = transfer of one
carbon unit or formate
-- its vitamin = folic acid
-- disease = megablastic anemia, birth defects
Coenzyme = biotin
a prosthetic group
-- use = carboxylations
-- its vitamin = biotin
Coenzyme = cyanocobalamin
-- use = methyl group transfer;
folate metabolism,
myelin synthesis
-- its vitamin =
cyanocobalamin
or vitamin B12
-- disease =
pernicious anemia
Coenzyme = lipoic acid
(reduced SH or oxidized form -S-S-)
prosthetic group (recall oxidized
pyruvate dehydrogenase)
-- use = redox reactions
-- its vitamin = lipoic acid
(humans probably produce
enough so it is not always
considered a vitamin) reduced
ascorbic acid or vitamin C
-- Not a coenzyme,
a cosubstrate
-- use = antioxidant
(aqueous phase),
Hydroxylations
(collagen)

-- disease = subacute scurvy, sore

and bleeding gums, loose teeth,
fatigue, lack of resistance to disease.
Scurvy itself is very unusual.
 Fat-Soluble Vitamins
The following fat-soluble vitamins are also
generally not coenzymes but are included here
for completion.
Vitamin A
-- its derivative retinal is the chromophore or light
absorbing factor in rhodopsin, our visual protein
-- use = vision, bone formation,differentiation
of epithelial cells, early development
-- disease = night blindness, sterility, skin lesions
Vitamin D
-- use = calcium and phosphate
absorption and metabolism
-- disease = rickets in children,
osteomalacia in adults
Vitamin E, tocopherol
-- use = antioxidant (lipid phase),
free radical trapping
-- disease = liver atrophy, hemolysis
of erythrocytes
Vitamin K
fat soluble
-- use = blood coagulation (needed for synthesis
of prothrombin), biosynthesis of
Ca binding proteins
-- disease = slow clotting time, excessive bleeding
 Reaction Rates and the
Transition State
Enzymes speed up reactions enormously.
To understand how they do this, examine
the concepts of activation energy &
the transition state.

In order to react, the molecules involved

are distorted, strained or forced to have
an unlikely electronic arrangement.
That is the molecules must pass through a
high energy state.
This high energy state is called
the transition state.
The energy required to achieve it is called
the activation energy for the reaction.
The higher the free
energy change for the
transition barrier,
the slower the
reaction rate.
Enzymes lower energy
barrier by forcing the
reacting molecules
through a different Enzyme
transition state.

This transition state

involves interactions
with the enzyme.
 Modes of Enzymatic
Enhancement of Rates
1) general acid and general base
catalysis-- good proton donors
& acceptors positioned just right.
2) covalent catalysis- unstable intermediate
3) metal ion catalysis
- electron donor or acceptor
4) electronic effects- “orbital steering”
of Koshland, steering aromatic groups
5) proximity and orientation
- close to reactive group
and aligned versus random
in solution chemistry.

6) conformational strain distortion

and induced fit
old idea, lock-and-key=
substrate fits active site
induced fit- enzyme changes its
conformation to accept the
transition state of substrate/product well.
Enzyme conformational
change works to distort
and strain substrate forcing
it into transition state.
Simultaneous
Koshland
 ENZYME KINETICS
Why study enzyme kinetics?
a) the precise scheduling of reactions
in a cell is important to the cell and our
understanding of its workings
b) enzyme mechanisms, e.g., the
number of kinetic steps and the detailed
chemistry can be learned (enzymology).
c) understanding enzyme function
leads to better drugs.
 Definition: rate of a reaction

For an enzyme acting on its substrate,

just as an ordinary chemical reaction,
the rate of the reaction depends on the
concentration of substrate, S.
A reaction leading to formation of
product is written:
S P

Rate = change in [P]/ change in time or

rate = v = Δ[P]/Δt
For a chemical reaction (as contrasted
to an enzymatically catalyzed one),
the rate is proportional to reactant [S].

A rate constant, k,
can be defined: rate

rate = v = Δ[P]/Δt
= k [S] [S]
- In contrast, found empirically for enzymes:

rate depends on [S] but

hyperbolic curve &
plateaus
rate
rate also depends
on the enzyme
concentration
[S]
 Michaelis-Menten Model
or Interpretation

E + S  E●S  E + P

where E●S is enzyme-substrate complex,

i.e., an intermediate complex.
rate stops increasing or plateaus
because the complex E●S becomes filled
at high [S]
Assigning rate constants to MM model:
k1 k3
E + S  E●S  E+ P
k2
From this kinetic scheme, a relationship
can be derived for the rate or velocity of
the reaction:
Michaelis-Menten Equation
Vmax[S]
V=
[S] + Km
gives hyperbolic curve on next slide.
Vmax, the maximum rate (plateau) is
k3 x [total enzyme]
Km =(k2 +k3)/k1, almost a binding constant
Vmax
Vmax is
approached Vmax/2
asymptotically
v or
rate Km
0
0 [S]
Unit of velocity is μmoles/min×mg protein
Km = [S], where the velocity v = Vmax /2,
is called the Michaelis constant.
Km is in units of concentration
Km good estimate
for the optimum Vmax
concentration
of substrate. Vmax/2
v
Km
[S]
A plot of v = Vmax[S]
[S] + Km
is not accurate enough to derive good
Km & Vmax.
Computer analysis is done.

Reciprocal Plot
A double reciprocal plot or
Lineweaver-Burk plot is linear and more
eye-appealing for presentation.
mathematically = “linear transformation”
Result is:

1/v = Km/Vmax●1/[S] + 1/Vmax

Looks like the linear y = m●x + b

m = slope b = intercept on y

1/v = Km/Vmax●1/[S] + 1/Vmax

“x”
Notice also intercept on “x” is -1/Km
 1/v
slope =
intercept Km/Vmax
= 1/Vmax
-1/Km
1/[S]
 Competitive Inhibition substrate
presented as double reciprocal plot
Model:
E + S  E●S  E + P
+
I inhibitor

E●I

I resembles S
I binds at active site reversibly
E●I cannot bind to S so no reaction
Competitive Inhibition
Vmax No I
+I
+more I

Km

In competitive inhibition, can always

add enough [S] to overcome inhibition.
 same Vmax
 Competitive inhibition
1/v
Double reciprocal plot + more I
+I
Same 1/v intercept,
same Vmax
1/Vmax
No I
Different slopes,
competitive
Inhibition changes apparent Km 1/[S]

Note: inhibition line always above no inhibition.

 Molecular interpretation
for competitive inhibition
competitive inhibitor binds to same site
as the substrate (competes).
its structure usually resembles
substrate or product.
While the inhibitor is bound, the enzyme
cannot bind substrate and no reaction
possible.

Many pharmaceutical agents are

competitive inhibitors so are many toxic
substances.
example: captopril
Blood pressure is regulated in kidney by renin,
a specific proteolytic enzyme, which acts on
angiotensinogen, the precursor for the active
regulator.
renin
angiotensinogen angiotensin I
asp-arg-val-tyr-ile-his-pro-phe-his-leu
converting enzyme
angiotensin II the active factor
O
peptide captopril HS-CH2-CH-C-N COOH
captopril is ACE inhibitor CH3 pro-like here
(angiotensin converting enzyme}
Finding useful inhibitors
Trial & error
Molecular
modeling
Testing enzyme
inhibition
Testing safety

Example:
HIV Protease is a dimer.
inhibitor is shown at active site.
interactions involve both subunits.
Noncompetitive Inhibition
substrate
E + S  E●S E + P
+ +
I I
 
E●I  E●S●l inhibitor

E●I and E●S●I not productive, depletes

E and E●S
Noncompetitive +I
Inhibition 1/v
No I

1/[S]
different slopes, different 1/v intercepts.
 Molecular Interpretation:
Inhibitor binds the enzyme somewhere
different from where the substrate binds.
So the inhibitor does not care whether
substrate is bound or not.
Inhibitor changes the conformation
of the enzyme at the active site so no
reaction is possible with inhibitor bound.

E●I and E●S●I not productive

 Irreversible Inhibition

reactive compounds

combine covalently to enzyme so as

to permanently inactivate it
(previous examples are all reversible)

almost all are very toxic

most bind to a functional group in

active site of enzyme to block that site
Example 1:
diisopropyl fluorophosphate (DFP) binds
covalently to serine in serine proteases
& acetylcholinesterase - tool for biochemists

sarin is a deadly nerve gas  Paralysis

O
Isopropyl-O-P-O-CH2- AChE
CH3
Example 2:
penicillin and related antibiotics bind
covalently to a peptidase involved in
cell wall synthesis in bacteria

Staphylococci, Streptococci sp. and others