Subject: Enzymology

Course Code: BT222

Topic: Enzymatic Catalysis
Submitted To: Dr Sarah Gul
Submitted By: Ayesha Hussain
Registration No: 598-FBAS/BSBT/F14
 Enzymatic Catalysis

Enzyme Catalysis
Enzyme Catalysis is a process that increase the rate at which a reaction approaches equilibrium.
Rate enhancement depends upon reduction of G‡ (activation barrier) relative to uncatalyzed
reaction.
Two related properties make enzyme amazingly powerful catalysts
1 – specificity of substrate binding
2 – optimal arrangement of catalytic groups
 Most enzymes are proteins, and most such processes are chemical reactions. Within the enzyme,
generally catalysis occurs at a localized site, called the active site.

Catalytic Mechanism
Enzymes enhance reactions in many ways
1. Acid-base catalysis
2. Covalent catalysis (mechanism depends on catalytic residue)
3. Metal ion catalysis
Additional mechanisms allow the enzyme substrate complex to lower its transition state energy
1. Electrostatic catalysis
 2. 5Proximity & orientation effects
3. Preferential binding of transition state complex

Acid-Base Catalysis
Acid catalysis involves partial proton transfer from a donor that lowers the free energy of the
transition state
Base catalysis involves partial proton abstraction from an acceptor that lowers the free energy of
the transition state
Biochemical reactions susceptible to acid-base catalysis include:
peptide and ester hydrolysis
tautomerizations
reactions of phosphate groups
additions to carbonyl groups
Typically involves Asp, Glu, His, Cys, Tyr & Lys residues
Most enzymes utilize concentrated acid-base catalysis mechanism.

Concentrated Acid-Base Catalysis

Promote reactions by increasing the strength of the nucleophile (proton abstraction) and/or the
stability of the leaving group (proton donation)
More RNase A
Enzyme kinetics, chemical modification studies and X-ray crystal structures indicate:
A) Two essential Histidine residues (H12 & H119)
B) Concerted general acid-base mechanism

Covalent Catalysis
Covalent catalysis involves the substrate forming a transient covalent bond with residues in the
enzyme active site or with a cofactor. This adds an additional covalent intermediate to the
reaction and helps to reduce the energy of later transition states of the reaction. The covalent
bond must be broken in the reaction at a later stage to regenerate the enzyme. This mechanism is
utilized by the catalytic triad of enzymes such as This mechanism is utilized by the catalytic
triad of enzymes such as proteases like chymotrypsin and trypsin.
Three stages in covalent catalysis
1) nucleophilic reaction between enzyme and substrate
2) electrophilic withdrawal of electrons from substrate
3) elimination reaction
Instead of lowering the activation energy for a reaction pathway, covalent catalysis provides an
alternative pathway for the reaction (via to the covalent intermediate) which differentiates it from
true catalysis.

Metal Ion Catalysis

A metal ion in the active site participates in catalysis by coordinating charge stabilization and
shielding. Because of a metal's positive charge, only negative charges can be stabilized through
metal ions. However, advantage of metal ions in biological catalysis is that they are not affected
by changes in pH. Metal ions can also act to ionize water by acting as a Lewis acid. Metal ions
may also be agents of oxidation and reduction.
Two classes of metal ion dependent enzymes
1) Metalloenzymes contain tightly bound transition metal ions (eg. Fe2+, Fe3+, Cu2+,
Zn2+, Mn2+, Co3+)
2) Metal-activated enzymes loosely bind metal ions (e.g. Alkali or alkaline metal
including Na+, K+, Mg2+ and Ca2+)
Metal ions enhance catalysis in three major ways
1)Binding to and orienting substrates for reaction e.g.
Mg2+ binding to ATP
2) Mediating redox reaction through changes in oxidation state e.g.
Reduction of O2 to H2O through electron transfer
3) Electrostatic stabilization or shielding of negative charges e.g. Mg2+ binding to ATP
Charge Stabilization
Metals can act as 'super acids'
Their role is similar to protons in acid catalyzed reactions
They are more effective than protons and given their [high] at neutral pH and charges > +1
Metals shield or reduce the effective charge on highly anionic substrate
It also facilitates nucleophile approach
Electrostatic Catalysis
Electrostatic catalysis occurs when the enzyme active site stabilizes the transition state of the
reaction by forming electrostatic interactions with the substrate. The electrostatic interactions can
be ionic, ionic-dipole, dipole-dipole, or hydrophobic interactions. Hydrogen bonding is one of
the most common electrostatic interactions formed in the active site.
Desolvation
Enzyme active sites can become devoid of water and mimic the reaction characteristics of the
gas phase. This can destabilize the polarized state of charged groups such as acids and bases.
Thus, the neutral form of these types of residues becomes the favored state. This is due to
significant alterations in the pKa of the active site residues within the nonpolar environment.
This can cause normally acidic residues such as glutamate to abstract a proton from histidine and
behave as a base.

Cofactor Catalysis

Cofactors are molecules that bind to enzymes and are required for the catalytic activity of the
enzyme.
  They can be divided into two major categories:
o metals 
o coenzymes
Metal cofactors that are commonly found in human enzymes include: iron, magnesium,
manganese, cobalt, copper, zinc, and molybdenum.
 Coenzymes are small organic molecules that are often derived from vitamins, which are
essential organic nutrients consumed within the diet. Coenzymes can bind loosely with the
enzyme and have the ability to bind and release from the active site, or they may be tight binding
and lack the ability to release easily from the enzyme.
Tight binding coenzymes are referred to as prosthetic groups.
Apoenzymes Enzymes that are not yet associated with a required cofactor are
called apoenzymes,
Holoenzymes whereas enzymes that are bound with their required cofactors are
called holoenzymes. 
Sometimes organic molecules and metals combine to form coenzymes, such as in the case of the
heme cofactor. Coordination of heme cofactors with their enzyme counterparts often involves
electrostatic interactions with histidine residues as shown in the succinate dehydrogenase
enzyme.

The Heme Cofactor. The family of heme cofactors contain an iron metal coordinated with a
porphyrin ring structure as shown in the left-hand panel within the structure of Heme B. In the
right-hand panel, Heme B is shown complexed with the succinate dehydrogenase enzyme from
the Krebs Cycle.

Examples of catalytic mechanism

Triose phosphate isomerase
Triose phosphate isomerase catalyzes the reversible interconversion of two triose phosphate
isomers

Trypsin
Trypsin is a serine protease that cleaves protein substrate after lysine or arginine residues using a
catalytic triad to perform covalent catalysis.

Aldolase
Aldose catalyzes the breakdown of fructose 1,6-biphosphate into glyceraldehyde 3-phosphate
and dihydroxyacetone phosphate.