Enzyme Catalysis
3/17/2003
General Properties of Enzymes
•Increased reaction rates sometimes 106 to 1012 increase
Enzymes do not change DG just the reaction rates.
•Milder reaction conditions
•Great reaction specificity
•Can be regulated
Substrate specificity
The non-covalent bonds and forces are maximized to
bind substrates with considerable specificity
•Van der Waals forces
•electrostatic bonds (ionic interactions)
•Hydrogen bonding
•Hydrophobic interaction
A+B
Substrates
enz
P+ Q
Products
Enzymes are Stereospecific
O

CH3CH 2OH  NAD  CH3CH  NADH  H
Yeast
Alcohol dehydrogenase

H
O
C
NH2
NAD+
CH3CD2OH +
+
N
Ox.
D
H
O
CH3C-D
O
C
+
N
NADD
Pro-R hydrogen gets pulled off
Yeast Alcohol dehydrogenase
NH2
Red.
OH
O
2. NADD + CH3-C-H
H
C
D
CH3
O
OH
3. CH3-C-D + NADH
D
C
H
CH3
If the other enantiomer is used, the D is not transferred
YADH is stereospecific for Pro-R abstraction
Both the Re and Si faced transfers yield identical
products. However, most reactions that have an Keq
for reduction >10-12 use the pro-R hydrogen while
those reactions with a Keq <10-10 use the pro-S
hydrogen. The reasons for this are still unclear
Specific residues help maintain
stereospecificity
Liver alcohol dehydrogenase makes a mistake
1 in 7 billion turnovers. Mutating Leu 182 to
Ala increases the mistake rate to 1 in 850,000.
This is a 8000 fold increase in the mistake rate,
This suggests that the stereospecificity is
helped by amino acid side chains.
Geometric specificity
Selective about identities of chemical groups
but
Enzymes are generally not molecule specific
There is a small range of related compounds that will
undergo binding or catalysis.
Similar shaped molecules can be highly toxic
H
O
C
NH2
N
+
N
N
CH3
Tobacco Nicotine
Because of this closeness in name
Nicotinic acid was renamed to niacin by the bread
manufactures
Coenzymes
Coenzymes: smaller molecules that aid in enzyme chemistry.
Enzymes can:
a. Carry out acid-base reactions
b. Transient covalent bonds
c. Charge-charge interactions
Enzymes can not do:
d. Oxidation -Reduction reactions
e. Carbon group transfers
Prosthetic group - permanently associated with an enzyme
or transiently associated.
Holoenzyme: catalytically active enzyme with cofactor.
Apoenzyme: Enzyme without its cofactor
Commom Coenzymes
Coenzyme
Reaction mediated
Biotin
Carboxylation
Cobalamin (B12)
Alkylation transfers
Coenzyme A
Acyl transfers
Flavin
Oxidation-Reduction
Lipoic acid
Acyl transfers
Nicotinamide
Oxidation-Reduction
Pyridoxal Phosphate
Amino group transfers
Tetrahydrofolate
One-carbon group transfers
Thiamine pyrophosphate
Aldehyde transfer
Vitamins are Coenzyme precursors
Vitamin
Coenzyme
Deficiency Disease
Biotin
Biocytin
Cobalamin (B12)
Cobalamin
Pernicious anemia
Folic acid
tetrahydrofolate
Neural tube defects
Megaloblastic anemia
Nicotinamide
Nicotinamide
Pellagra
Pantothenate
Coenzyme A
Not observed
Pyridoxine (B6)
Pyridoxal phosphate
Not observed
Riboflavin (B2)
Flavin
Not observed
Thiamine (B1)
Thiamine pyrophosphate
Beriberi
not observed
These are water soluble vitamins. The Fat soluble
vitamins are vitamins A and D.
Humans can not synthesize these and relay on their
presence in our diets. Those who have an unbalanced diet
may not be receiving a sufficient supply.
Niacin (niacinamide) deficiency leads to pellagra
characterized by diarrhea, dermatitis and dementia.
Pellagra was endemic is Southern United States in the
early 20th century. Niacin can be synthesized from the
essential amino acid, tryptophan. A corn diet prevalent at
the time restricted the absorption of tryptophan causing a
deficiency. Treatment of corn with base could release the
tryptophan (Mexican Indians treated corn with Ca(OH)2
before making tortillas!)
Regulation of Enzymatic Activity
There are two general ways to control enzymatic activity.
1. Control the amount or availability of the enzyme.
2. Control or regulate the enzymes catalytic activity.
Each topic can be subdivided into many different
categories.
Enzyme amounts in a cell depend upon the rate in which it
is synthesized and the rate it is degraded. Synthesis rates
can be transcriptionally or translationally controlled.
Degradation rates of proteins are also controlled.
However, We will be focusing on the regulation of
enzymatic activity.
The catalytic activity of an enzyme can be altered
either positively (increasing activity) or negatively
(decreasing activity) through conformational
alterations or structural (covalent) modifications.
Examples already encountered is oxygen, carbon
dioxide, or BPG binding to hemoglobin.
Also, substrate binding to the enzyme may also be
modified by small molecule effectors changing its
catalytic site.
Protein phosphorylation of Ser residues can activate
or deactivate enzymes. These are generally
hormonally controlled to ensure a concerted effect on
all tissues and cells.
Aspartate Transcarbamoylase:
the first step in pyrimidine biosynthesis.
O
O
-
NH2
O
CH2
OPO3--
ATCase
+
H3N+
Carbamoyl
phosphate
O
C
C
C
O-
C
H
CH2
NH2
H
PO
2
4
+
C
COO
Aspartate
-
O
N
H
C
H
COO-
N-Carbamoyl aspartate
This enzyme is controlled by Allosteric regulation and
Feedback inhibition
Notice the S shaped curve (pink) cooperative binding of aspartate
Positively homotropic cooperative binding
Hetertropically inhibited by CTP
Hetertropically activated by ATP
Feedback inhibition
Where the product of
a metabolic pathway
inhibits is own
synthesis at the
beginning or first
committed step in the
pathway
CTP is the product of this pathway and it is also a
precursor for the synthesis of DNA and RNA (nucleic
acids). The rapid synthesis of DNA and/or RNA
depletes the CTP pool in the cell, causing CTP to be
released from ATCase and increasing its activity. When
the activity of ATCase is greater than the need for CTP,
CTP concentrations rise rapidly and rebinds to the
enzyme to inhibit the activity.
ATP activates ATCase. Purines and Pyrimidines are
needed in equal amounts. When ATP concentrations
are greater than CTP, ATP binds to ATCase activating
the enzyme until the levels of ATP and CTP are about
the same.
Enzymatic catalysis and mechanisms
•A. Acid - Base catalysis
•B. Covalent catalysis
•C. Metal ion aided catalysis
•D. Electrostatic interactions
•E. Orientation and Proximity effects
•F. Transition state binding
General Acid Base
Rate increase by partial proton abstraction by a
Bronsted base or
Rate increase by partial proton donation by a
Bronsted Acid
Mutarotation of glucose by acid and base catalysts
d   D  glucose
v
 k obs  D  glucose
dt
The reaction can be followed by observation of the optical activity
change
Kobs = apparent first order kinetics but increases with
increased concentrations of acid and base.
The acid HA donates a
proton to ring oxygen,
while the base abstracts a
proton from the OH on
carbon 1. To form the
linear form. The cycle
reverses itself after
attacking the carbonyl
from the other side.
This compound does not undergo mutarotation in
aprotic solvents. Aprotic solvents have no acid or
base groups i.e. Dimethyl sulfoxide or dimethyl
formamide. Yet the reaction is catalyzed by
phenol, a weak acid and pyridine, a weak base
v = k[phenol][pyridine][TM--D-glucose]
The reaction can be catalyzed by the addition of
-Pyridone as follows
v=k'[-pyridone][TM-a-D-glucose]
k' = 7000M x k or 1M -pyridone equals
[phenol]=70M and [pyridine]=100M
Many biochemical reactions require acid
base catalysis
•Hydrolysis of peptides
•Reactions with Phosphate groups
•Tautomerizations
•Additions to carboxyl groups
Asp, Glu, His, Cys, Tyr, and Lys have pK’s near
physiological pH and can assist in general acid-base
catalysis.
Enzymes arrange several catalytic groups about the
substrate to make a concerted catalysis a common
mechanism.
RNase uses a acid base mechanism
Two histidine residues catalyze the
reaction. Residue His 12 is deprotonated
and acts as a general base by abstracting
a proton from the 2' OH.
His 119 is protonated and acts as a
general acid catalysis by donating a
proton to the phosphate group.
The second step of the catalysis His 12
reprotonates the 2'OH and His 119 reacts
with water to abstract a proton and the
resulting OH- is added to the phosphate.
This mechanism results in the hydrolysis
of the RNA phosphate linkage.
Covalent catalysis
Covalent catalysis involves the formation of a
transient covalent bond between the catalyst and
the substrate
Catalysis has both an nucleophilic and an electrophilic stage
1 Nucleophilic reaction forms the covalent bond
2 Withdrawal of electrons by the now electrophilic catalyst
3 Elimination of the catalyst (almost the reverse of step 1)
Depending on the rate limiting step a covalent
catalytic reaction can be either elecrophilic or
nucleophilic. Decarboxylation by primary amines
are electrophilic because the nucleophilic step of
Schiff base formation is very fast.
Nucleophilicity is related to the basicity but
instead of abstracting a proton it attacks and forms
a covalent bond.
Lysines are common in formation of schiff bases while
thiols and imidazoles acids and hydroxyls also have
properties that make good covalent catalysts
Thiamine pyrophosphate and pyridoxal phosphate
also show covalent catalysis
Metal ion catalysts
One-third of all known enzymes needs metal ions to work!!
1. Metalloenzymes: contain tightly bound metal ions: I.e.
Fe++, Fe+++, Cu++, Zn++, Mn++, or Co++.
2. Metal-activated enzymes- loosely bind ions Na+, K+,
Mg++, or Ca++.
They participate in one of three ways:
a. They bind substrates to orient then for catalysis
b. Through redox reactions gain or loss of electrons.
c. electrostatic stabilization or negative charge
shielding
Charge stabilization by metal ions
Metal ions are effective
catalysts because unlike
protons the can be
present at higher
concentrations at
neutral pH and have
charges greater than 1.
Metal ions can ionize water at higher
concentrations
The charge on a metal ion makes a bound water more
acidic than free H2O and is a source of HO- ions even
below pH 7.0
NH3 5 Co3 H2O  NH3 5 Co3 OH-  H
The resultant metal bound OH- is a potent nucleophile
Carbonic Anhydrase

CO 2  H 2O  HCO3  H
Charge shielding
Proximity and orientation effects


d p  NO2O
 k1imidizolep  NO2Ac  k1 p  NO2Ac
dt
k'1 = 0.0018s-1 when [imidazole] = 1M
When the phenyl acetate form is used
k2 = 0.043 or 24k'1
Proximity effects lead to relatively small rate
enhancement!
•Reactants are about the same size as water molecules
(approximation)
•Each species has 12 nearest neighbors (packed spheres)
•Reactions only occur between molecules in contact
•Reactant conc. Is low so only one can be in contact at a
time
k1
A  B  A
dA B
B v
 k1AB  k 2 A, Bpair
dt
12AB
A, Bpairs 
55.5M
55.5
v  k1
A, Bpairs  4.6k1A, Bpairs
12
Only a 4.6 rate enhancement but molecular motions
if slowed down leads to a decrease in entropy and
rate enhancements.
Molecules are not as reactive in all directions and
many require proper orientation to react. Increases
in rates of 100 fold can be achieved by holding the
molecules in their proper orientation for reaction.
Preferential transition state binding
Binding to the transition state with greater affinity to
either the product or reactants.
RACK MECHANISM
Strain promotes faster rates
The strained reaction more closely resembles the
transition state and interactions that preferentially
bind to the transition state will have faster rates
kN
S 
 P
kE
ES 
 EP
kN for uncatalyzed reaction
and
kE for catalyzed reaction
K N‡
‡
E  S  S  E  P  E
 KR
‡
KE
 KT
‡
ES  ES

ES
KR 
ES

ES 
KE 
ES
‡
‡


EP

E

S

‡
K N‡ 

ES‡ 
KT 

E S 
‡
ES 
K T SES  K E
 ‡

‡
K R S ES K N
‡
‡
 k BT  ‡  k BT  ‡
v N  k N S  
S   
K N S
 h 
 h 
 k BT  ‡  k BT  ‡
vE  
ES   
K E ES
 h 
 h 
‡
k E K E KT

‡
k N K N KR
Preferential transition state binding
The more tightly an enzyme binds its reaction’s
transition state (KT) relative to the substrate
(KR) , the greater the rate of the catalyzed
reaction (kE) relative to the uncatalyzed
reaction (kN)
Catalysis results from the preferred binding
and therefore the stabilization of the transition
state (S ‡) relative to that of the substrate (S).
DGN  DGE  
kE

RT 
 exp
kN
The enzyme binding
of a transition state
(ES‡ ) by two
hydrogen bonds that
cannot form in the
Michaelis Complex
(ES) should result in
a rate enhancement
of 106 based on this
effect alone
106 rate enhancement
requires a 106 higher
affinity which is 34.2
kJ/mol
Transition state analogues are competitive
inhibitors