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Chapter 14

Enzymatic Catalysis
Part I

Read based on what


is covered in class

Although the catalytic properties


of enzymes may seem almost
magical, it is simply chemistry
the breaking and making of
bondsthat gives enzymes their
prowess. Garrett and Grisham
ATP

G1P

ADPGlucose Pyrophosphorylase with bound substrates

Types of Catalytic Mechanisms:


1. Proximity and Orientation effects
2. Preferential binding of the transition state
complex
3. General acid-base catalysis
4. Metal ion catalysis
5. Electrostatic catalysis
6. Covalent catalysis

Mechanism 1:
Proximity and Orientation effects

Binding to Enzyme active site positions substrates


for a productive interaction
No proper relative
orientation between
molecules
(no reaction)
Productive relative
orientation between
molecules
(reaction can occur)

Much more important than proximity aloneWHY?

Catalysis through orientation effects

In A, imidazole position can vary


In B, imidazole is position fixed and ready to attack carboxyl

K1 = 0.0018 s-1

B
K1 = 0.043 s-1

Organic chemistry reaction rates can be increased by


5
limiting rotional/translational freedom of reactants.
Enzymes mimic this in active site.

Deviating by as
little as 10 degrees
from optimal
approach angle
can decrease rate
by 100-fold!
Reaction rates are greatly
increased by freezing out
relative translational and
rotational motions of
reactants (decreasing
entropy)increased
fraction is in conformation
to enter transition state

Relative rate constant


1
~1 x 103
~2.3 x 105
~8 x 107

Mechanism 2:
Transition State Stabilization

If enzyme active site binds the transition state better than original
substrate, it can bend substrate into its transition state
Stick must be bent
(transition state) before it
gives broken products

If enzyme is perfect
complement to substrate,
no reaction

Enzyme pocket
complementary to transition
state helps destabilize
substrate, increase rate

**The Tight Binding of Transition State Analogs 7


provides Support for the Transition State Theory

Planar Transition state

Planar Transition state

Planar Transition state ANALOGS

Transition state ANALOG

*160 X tighter binding than substrate

Transition state analogs are stable


molecules that are chemically and
structurally similar to the transition
statebind tighter than S or
competitive Inhibitor

Transition state ANALOG

Transition State Stabilization idea can be used to


create ANTIBODIES WITH ENZYME ACTIVITY
(Catalytic Antibodies or Abzymes)
Example: you can create an
antibody that catalyzes
trypsin-like protease activity

How?

To make a catalytic antibody, immunize


mouse with a TRANSITION STATE
ANALOG (rather than substrate)

A Biotech Application of Transition State Theory 10


has been the Generation of Catalytic Antibodies
Abzymes are generated by raising antibodies to
compounds (coupled to carrier protein) designed to
mimic the transition state of the reaction
Abzymes have been shown to catalyze (up to 108
faster) a vast array of reactionseven reactions for
which there are no known natural enzymes-- with
specificity and stereoselectivity
Antigen binding sites of antibodies contain
ionized amino acids, activities pH dependent;
catalytic mechanisms are similar to real
enzymesfor example, an Abzyme with serine
protease activity was found to have a His and Ser in
the active site (we will be discussing the mechanism
of serine proteases in detail)

The first Abzymes created


catalyzed the hydrolysis of esters
Antibodies were
raised against
compounds that
mimic the
transition state of
ester hydrolysis

transition state
mimic was a
tetrahedral anionic
phosphonate

An exciting application is the generation of extremely


specific proteases

11

3 more examples of Abzymes made by using a 12


transition state analog as antigen (immunizing agent)
Adapted from Y. Xu et al Biorg. Med. Chem. 12 (2004) 5247-5268

antigen

Amide hydrolysis is difficult


amine is a poor leaving group;
above induced with
phosphoamidate; Arg involved in
t-state, His- Nu

antigen

antigen

Sulfonamide mimic of
tetrahedral intermediate
(and related transition state
of a distorted amide
hydrolysis

Diels-Alderrare reaction in
natureproceeds by an
entropically disfavored, highly
organized t-state; binding pocket
utilizes Van der Waals,
stacking, H-bonds

Mechanism 3: Acid-Base Catalysis


The active site of some enzymes contain amino
acid functional groups, which can participate in
acid-base catalysis

13

You should know the


general pK range of
these groups

From Lehninger
Principles of Biochemistry

Acid-Base Catalysis

(ex 1. catalysis of keto-enol tautomerization)


Carbanion like
High free energy

Uncatalyzed
slow
General acid
catalyzed

General base
catalyzed

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Acid-Base Catalysis
(ex 2: acid-base catalsis of Mutarotation)

The mutarotation rate increases with the concentrations


of general acids and bases

15

Acid-Base Catalysis and pH Profiles


Group acts as
general base
Group acts as
general acid
Involvement of
two groupslow
pK: base, high
pK: acid
There are two active site Asp groups in HIV-1 Protease

16

Acid-Base Catalysis: DETAILED


EXAMPLE (please know this)
(bovine pancreatic RNase S)
RNase S is the catalytically
active form of RNase A

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RNase A

- Digestive enzyme
- Secreted by the
pancreas into the small
intestine
- Functions to hydrolyze
RNA to its component
nucleotides

Non-hydrolyzable substrate analog is bound in the active site

- RNase A reaction
incorporates general
acid-base catalysis

The RNase A
mechanism

RNase A has 2 essential


His (His 12 & His 119)
His 12 (acting as a base
abstracts H+) from 2-OH
group
Promotes nucleophilic
attack on adjacent P atom
His-119 acting as acid
protonates the leaving
group

Intermediate

18

His 12 (acting as an acid)


& His 119 as a general
base

Mechanism 4:
Metal Ion Catalysis

19

Nearly 1/3 of all known enzymes require the presence of


metal ions for catalysis
A. Metalloenzymes (tightly bound metal ions [Fe, Cu, Zn,
Co])
B. Metal-activated enzymes (loosely bound from solution
[Na, K, Mg, Ca])
Metal ions participate in 3 different ways:
1) Binding substrates to orient them properly
2) Mediating redox reactions (via reversible changes in the
metal ions oxidation state)
3) Electrostatically stabilizing or shielding of negative
charges

Metal Ion Catalysis


Electrostatic Stabilization
The decarboxylation of
dimethyloxaloacetate (nonenzymatic)

20
Nucleophillic Catalysis via
Water Ionization

Metal ion stabilizes


the developing
enolate ion t-state
Base catalysis by His leads to OHformation (H-bonded network)
Most enzymes that
decarboxylate OAA
require metal ion
Charge Shielding

Actual substrate of kinase is Mg-ATP;


facilitates nucleophillic attack

Zn bound OH- ion Nu attacks enzyme


bound CO2, converting it to HCO3-

Mechanism 5:
Electrostatic
Catalysis
Electrostatic
Catalysis

-Can utilize acid-base and Metal ion catalysis

21

Mechanism 6:
Covalent Catalysis
General :
BX + Y

BY + X

Enzymatic version:
BX + E

E:B + Y

E + BY

X
If enzymatic pathway is faster, acceptor group on
enzyme must be a better attacking group than Y
and a better leaving group than X (many have
ping-pong mechanisms)

22

Covalent Catalysis continued


Rate enhancement via transient formation of
E-S covalent bond
Side chains in proteins have a variety of
nucleophillic centers for catalysisamines,
carboxylates, hydroxyls, imidazoles, thiol
groups (also coenzymes such as thiamine
pyrophosphate and pyridoxal phosphate)
Groups can attack electrophillic centers of
substrates, forming covalently bonded
intermediates
Covalent intermediate is then attacked by
water or a second substrate giving product

23

24

We will be discussing chymotrypsin in detail

Decarboxylation of acetoacetate

25
Uncatalyzed reaction

Catalyzed by
primary amines

catalyzed reaction

Biologically important nucleophilic


and electrophilic groups

Contain an edeficient atom

26

Entropy Loss / Destabilization of the ES complex: 27


Substrate binding to enzyme can remove waters of
solvation, making substrate more reactive
Formation of ES complex
results in a loss of entropy
(ES complex is more
ordered)
S typically lose water of
hydration; desolvation
raises energy, making
ES more reactive
Charged groups may be
forced to interact with likecharges (electrostatic
destabilization