ENZYMES

1
ENZYMES Definitions-- ?Chem
ical reactions in cells require specific
catalysis. ?Enzymes are proteins which
perform this function. ?Metabolite acted upon is
called the enzymes substrate.
2
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.
3
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
4
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
5
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.)
6
Class 1. Oxidoreductases- catalyze
redox processes Example RCH2-OH ?
RCHO 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
7
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 ---CC--- ?
---C--C---
8
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
ADPPi O -OOC-C-CH3 CO2
-OOC-C-CH2-COO- pyruvate
oxaloacetate
enzyme pyruvate carboxylase
9
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.
10
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
11
Coenzyme thiamine pyrophosphate (TPP)
? use decarboxylation and transketolation ?its
vitamin thiamine or vitamin B1, contains
pyrimidine and thiazole. ?disease beri-beri,
Wernikes disease. peripheral nerves, muscle
cramps, numbness
12
Coenzyme flavin mononucleotide (FMN), flavin
adenine dinucleotide (FAD) both act as
prosthetic groups -- use redox reactions -- its
vitamin riboflavin or B2
riboflavin
13
nicotinamide-adenine dinucleotide
(NAD), nicotinamide-adenine dinucleotide
phosphate (NADP) -- use redox reactions with H
transfer -- its vitamin niacin or B3
nicotinamide nicotinic acid -- disease
pellagra, skin lesions, swollen tongue,
nervous/mental disorders
Coenzyme
P
14
Coenzyme pyrodoxal phosphate -- use
decarboxylations, transaminations and
racemases -- its vitamin pyridoxine, or vitamin
B6
15
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
16
Coenzyme folate or tetrahydrofolate (the
reduced form) -- use transfer of one carbon
unit or formate -- its vitamin folic acid --
disease megablastic anemia, birth defects
17
Coenzyme biotin a prosthetic group -- use
carboxylations -- its vitamin biotin
18
Coenzyme cyanocobalamin -- use methyl group
transfer folate metabolism, myelin synthesis --
its vitamin cyanocobalamin or vitamin B12 --
disease pernicious anemia
19
Coenzyme lipoic acid (reduced SH or oxidized
form -S-S-) prosthetic group (recall pyruvate
dehydrogenase) -- use redox reactions -- its
vitamin lipoic acid (humans probably
produce enough so it is not always considered a
vitamin) reduced
oxidized
20
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.
21
Fat-Soluble Vitamins
The following fat-soluble vitamins are
also generally not coenzymes but are included
here for completion.
22
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
23
Vitamin D -- use calcium and phosphate absorptio
n and metabolism -- disease rickets in
children, osteomalacia in adults
24
Vitamin E, tocopherol -- use antioxidant (lipid
phase), free radical trapping -- disease liver
atrophy, hemolysis of erythrocytes
25
Vitamin K fat soluble -- use blood coagulation
(needed for synthesis of prothrombin),
biosynthesis of Ca binding proteins -- disease
slow clotting time, excessive bleeding
26
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.
27
?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.
28
Enzymes lower energy barrier by forcing
the reacting molecules through a
different transition state. This transition
state involves interactions with the enzyme.
Enzyme
29
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
30
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
31
?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
32
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.
33
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 ?P/?t
34
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 v
?P/?t k S
rate
S
35
- In contrast, found empirically for
enzymes ?rate depends on S but ?hyperbolic
curve ?plateaus ?rate also depends on the
enzyme concentration
rate
S
36
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
37
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 VmaxS
S Km gives hyperbolic curve on next slide.
V
38
Vmax, the maximum rate (plateau) is k3 x
total enzyme Km (k2 k3)/k1, almost a binding
constant
Vmax
Vmax is approached asymptotically
Vmax/2
v or rate 0
Km
S
0
Unit of velocity is µmoles/minmg protein
39
?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 concentration of substrate.
Vmax
Vmax/2
v
Km
S
40
?A plot of v VmaxS
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. ?mathematical
ly linear transformation
41
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
42
1/v
x x x x x x x
slope Km/Vmax
intercept 1/Vmax
-1/Km
1/S
43
Competitive Inhibition presented as double
reciprocal plot Model E S ? E?S ? E P
I ? E?I I resembles S I binds at active
site reversibly E?I cannot bind to S so no
reaction
substrate
inhibitor
44
Competitive Inhibition
Vmax
No I
I
more I
Km
In competitive inhibition, can always add enough
S to overcome inhibition.
? same Vmax
45
Competitive inhibition
1/v
Double reciprocal plot
more I
I
?Same 1/v intercept, same Vmax ?Different
slopes, competitive Inhibition changes apparent
Km ?Note inhibition line always above no
inhibition.
1/Vmax
No I
1/S
46
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.
47
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
48
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.
49
Noncompetitive Inhibition E S ? E?S? E P
I I ? ? E?I ? E?S?l
substrate
inhibitor
E?I and E?S?I not productive, depletes E and E?S
50
Noncompetitive Inhibition
I
1/v
No I
1/S
different slopes, different 1/v intercepts.
51
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
52
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
53
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-CH
2- AChE CH3
54
Example 2 penicillin and related antibiotics
bind covalently to a peptidase involved in cell
wall synthesis in bacteria Staphylococci,
Streptococci sp. and others