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ENZYME MECHANISMS
Today we will consider aspects of the spectacular ability of enzymes to catalyze
chemical reactions. We will cover:
1) General Aspects. Enzymes catalyze reactions by lowering the activation
energy barrier for the reaction and often providing alternate pathways for a
reaction to occur. We will discuss four general categories of catalytic mechanisms
including a) transition state stabilization, b) general acid-base catalysis, c)
covalent catalysis and d) metal ion catalysis.
2) Classifications of Enzymes. Six different general classes of enzymes will be
discussed in order to provide a conceptual framework for enzymes that will be
covered in the course.
3) Proteases. As examples of enzymes with important medical consequences,
we will discuss the properties and mechanisms of proteases, including four major
types of proteases. We will finish with a discussion of the mechanistic aspects of
catalysis by serine proteases.
Reading: Lippincott Chapter 5, sections I - IV
I. General Aspects of enzymatic reactions and transition states
Consider a reaction A + B  P + Q
A+B
K‡
‡
k'

P+Q
where A + B react through transition state, X‡, to form products P + Q. K‡ is the
equilibrium constant between A + B and X‡ and k' is the rate constant for
conversion of X‡ to P + Q.
‡
G ‡
G
A+B
G reaction
P+Q
Reaction coordinate
The minimum energy pathway of the reaction
is shown in the reaction coordinate, or
transition state diagram, at left. Chemical
conversion of A + B to P + Q proceeds
through a transition state ‡ which is the
least stable (least probable, highest free
energy) species along the pathway.
Molecules that achieve the activation energy,
G‡ , can go on to react while molecules that
fail to achieve the transition state fall back to
the ground state.
The transition state, X‡, is metastable with only a transient existence. The less stable
the transition state, the more difficult it is for a reaction to proceed.
The smaller the difference in free energy of the reactants and the transition state, the faster the
reaction proceeds. Enzymatic rate accelerations are achieved by lowering the activation
barrier between reactants and the transition state, thereby increasing the fraction of
reactants able to achieve the transition state. Enzymes catalyze reactions by either
stabilizing the normal transition state or providing an alternative pathway from
reactants to products.
a) Transition state
stabilization by induced fit
b) Acid-base catalysis
c) Covalent Catalysis
d) Metal ion catalysis
a) Transition state stabilization
Imaginary enzyme ("stickase") designed to catalyze "cleavage"
(breaking) of a metal stick ("magnetic" interactions, red dashed lines,
represent noncovalent interactions between enzyme and substrate and
between enzyme and transition state ‡). Metal stick must be bent, a
"high energy state", before it can be broken, so "transition state" is
bent stick.
(Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000)
No Enzyme
No catalysis is obtained by just binding substrate tightly!
b) General acid-base
catalysis:
Often used in the hydrolysis of
ester/ peptide bonds, phosphate
group reactions, addition to
carbonyl groups, etc.
What side-chains can act donate
or accept protons?
Amino Acid
Aspartic acid
pK a
3.90
-COOH
COO -
O
H C CH 2 C
ONH 3+
-COOH
COO -
An enzyme avoids unstable
charged intermediates in
reaction (which would have high
free energies) by having groups
appropriately located to:
Glutamic acid
4.07
H C CH 2
NH 3+
CH 2 C
O-
COO Histidine
6.04
H C CH
NH 3+
imidazole
N
2
N
COO Cysteine
8.33
H C CH
NH 3+
donate a proton (act as a
general acid), or
sulfhydryl
2
SH
phenol
COO Tyrosine
accept a proton (abstract a
proton, act as a general base)
O
10.13
H C CH
NH 3+
OH
2
-amino
COO Lysine
10.79
H C CH 2
NH 3+
CH 2
CH2 NH +
3
c) Covalent catalysis (also sometimes called nucleophilic catalysis):
(transient formation of a catalyst-substrate covalent bond)
Nucleophilic form
Biologically important
nucleophilic groups:
Hydroxyl group
R-OH
R-O:
Sulfhydryl group
R-SH
R-S: -
+ H+
Amino group
R-NH3+
R-NH2
+ H+
R
Imidazole group
HN + NH
+ H+
R
HN
N:
+ H+
Biologically important
electrophiles:
H+
Protons
Mn+
Metal Ions
C=O
Carbonyl carbon
R-NH2
+ C=O
Adapted from Voet & Voet, Biochemistry
d) Metal ion catalysis:
Metal ions are often used for one or more of the following:
*
binding substrates in the proper orientation
*
mediating oxidation-reduction reactions
*
electrostatically stabilizing or shielding negative
charges (electrostatic catalysis)
Metalloenzymes contain tightly bound metal ions: (usually Fe+2,
Fe+3, Cu+2, Zn+2, or Mn+2)
Metal-activated enzymes contain loosely bound metal ions:
(usually Na+, K+, Mg+2, or Ca+2)
Which of the following mechanisms is not used by enzymes
for catalysis?
A. Acid base catalysis
B. Induced fit of intermediate
C. Providing complementary electrostatics
D. Binding of metal ions
E. Destabilizing the transition state
Which of the following residue side-chains could not
contribute to acid-base catalysis?
A. Lysine
B. Glutamate
C. Histidine
D. Phenylalanine
E. Tyrosine
II. Enzyme Classes
Enzymes have been grouped into 6 general classes by “The Enzyme Commission”. These
include 1) Oxidoreductases, 2) Transferases, 3) Hydrolases, 4) Lyases, 5) Isomerases
and 6) Ligases. Since such classifications can aid recognition of enzymes, we will briefly
discuss each of these groups and provide examples.
1) Oxidoreductases: This is a very broad class of enzymes that catalyze the many
oxidation-reduction reactions found in biochemical pathways. Oxidoreductases catalyze
reactions in which at least one substrate gains electrons, becoming reduced, and another
loses electrons, becoming oxidized.
An important subset of oxidoreductases are the dehydrogenases
that accept and donate electrons as
hydride ions (H:-) or hydrogen
atoms often using cofactors such as
NAD+/NADH as an electron donor
or acceptor. An example is Lactate
Dehydrogenase (LDH) (right).
LDH
R
NAD+
R
NADH
(From “Mark’s Basic Medical Biochemistry – A clinical approach”)
2) Transferases: This class of enzymes catalyze
the transfer of a specific functional group between
molecules. Important subsets of transferases include
a) Kinases that transfer phosphate groups,
usually from ATP to another molecule (such as
hexokinase and glucokinase, that phosphorylate
glucose and protein kinases that phosphorylate
protein hydroyxl groups), b) Aminotransferases (see
right) that transfer amino groups that are important in
amino acid metabolism, c) Acyltransferases that
transfer fatty acyl groups and d)
Glycosyltransferases, which transfer carbohydrate
residues.
Aminotransferase reaction in which
Pyridoxal phosphate (PLP) is used as a
cofactor. (From “Mark’s Basic Medical
Biochemistry – A clinical approach”)
3) Hydrolases:
Hydrolysis reactions refer to the cleavage of bonds by the addition of a
water molecule. A very important class of hydrolases are the proteases involved in
cleaving peptide bonds, as we will be discussing shortly
4) Lyases: This class refers to those enzymes involved in cleaving bonds by means
other than hydrolysis or oxidation. Examples include aldolases (such as fructose
diphosphate aldolase, which is involved in glycolysis) and thiolases (such as ketoacyl-CoA thiolase involved in the breakdown of fatty acids). Lyases also
include enzymes involved in elimination of groups from two adjacent carbon atoms
to form double bonds.
In glycolysis, fructose 1,6bisphosphate aldolase cleaves a
carbon-carbon bond in fructose
1,6-bisphosphate. (From “Mark’s
Basic Medical Biochemistry – A
clinical approach”)
5) Isomerases: At many stages of metabolism,
rearrangement of the atoms of a molecule is
required to create an isomer of the starting
compound. Enzymes generally catalyzing the
rearrangement of the bond structure are called
isomerases, while those specifically catalyzing
the movement of a phosphate from one group to
another are known as mutases. For example,
triose phosphate isomerase (right) catalyzes the
interconversion between dihydroxyacetone
phosphate and D-glyceraldehyde 3-phosphate,
which is essential for continuing glycolysis
following splitting of six carbon sugars into two
three carbon sugars by fructose diphosphate
aldolase
(From “Mark’s Basic Medical
Biochemistry – A clinical approach”)
6) Ligases: Ligases are involved in synthesizing bonds between carbon atoms and
carbon, nitrogen, oxygen or sulfur atoms in reactions that are coupled to the cleavage of
the high energy phosphate of ATP or another nucleotide. Pyruvate carboxylase, a key
enzyme in gluconeogenesis, is one important ligase in metabolism.
Proteases cleave peptide bonds by the addition of water. To
what class of enzymes do they belong?
A. Oxidoreductases
B. Ligases
C. Hydrolases
D. Lyases
E. Isomerases
15
III. Proteases - Enzymes that specifically cut other proteins and
are important in regulation
(Ser, Cys or Thr)
(Asp or Glu)
(usually His)
(metal)
Protease classification
1) Cysteine Proteases
2) Aspartyl Proteases
3) Metalloproteases
4) Serine Proteases
1) Cysteine Proteases
MECHANISM: In cysteine proteinases, catalysis proceeds
through the formation of a covalent intermediate and involves a
cysteine and a histidine residue. The essential Cys and His play the
same role as Ser and His respectively in Serine proteases as
discussed later. The nucleophile is a thiolate ion that is stabilized
through the formation of an ion pair with neighbouring
imidazolium group of His. The attacking nucleophile is the
thiolate-imidazolium ion pair in both steps.
EXAMPLES:
Medically interesting cysteine proteases include:
- mammalian enzymes such as cathepsins B and L, which are involved in cancer growth
and metastasis, and cathepsin K, which is important for bone degradation an osteoporosis.
- Cruzipain and cruzain from Trypanosoma cruzi, which cause Chagas' disease, a
permanent infection that affects more than 25 million people annually in South America and
causes more than 45,000 deaths per year, and falcipain, from Plasmodium falciparum,
which causes malaria.
- Caspases, which are key mediators of apoptosis.
2) Aspartyl Proteases
MECHANISM: In contrast to cysteine (and serine) proteases,
catalysis by aspartic proteinases do not involve a covalent
intermediate, even though a tetrahedral intermediate is
transiently formed. Rather, nucleophilic attack is achieved by
two simultaneous proton transfers: one from a water molecule to
one of the two carboxyl groups and a second one from the
carbonyl oxygen of the substrate with the concurrent CO-NH
bond cleavage. This general acid-base catalysis, which may be
called a "push-pull" mechanism leads to the formation of a non
covalent neutral tetrahedral intermediate
EXAMPLES:
Plasmepsin, which is produced in the parasite that causes malaria, is part of a closely related
group of enzymes known as aspartyl proteases. Plasmepsin is believed to play a key role in the
digestion of the human host's hemoglobin, the major nutrient source for the parasite.
HIV protease permits viral maturation.
BACE, an aspartyl protease involved in the amyloid peptide generation of Alzheimer's
disease.
3) Metalloproteases (Zn)
MECHANISM: Many enzymes contain the sequence HEXXH,
which provides two histidine ligands for binding of zinc. A third Zn
ligand is either a glutamic acid (thermolysin, neprilysin, alanyl
aminopeptidase) or a histidine (astacin, serralysin). Other families
exhibit a distinct mode of binding of a Zn ion. The catalytic
mechanism involves formation of a non covalent tetrahedral
intermediate after the attack of a zinc-bound water molecule on the
carbonyl group of the scissile bond. This intermediate can be further
decomposed by transfer of the glutamic acid proton to the leaving
group.
EXAMPLES:
Matrix metalloproteinases (MMPs) are a family of enzymes that are responsible for the
degradation of extracellular matrix components such as collagen, laminin and proteoglycans.
These enzymes are involved in normal physiological processes such as embryogenesis and tissue
remodeling and may play an important role in arthritis, periodontitis, and metastasis.
ACE is a metalloprotease that catalyses the conversion of angiotensin I into angiotensin II, which
leads to vasoconstriction. ACE inhibitors, were originally used as antihypertensives, but have
significantly improved the treatment of other cardiovascular diseases and are now used to treat
heart failure and even prevent heart attacks in at-risk patients.
4) Serine Proteases
MECHANISM:
The key active site groups are Ser, His and Asp. These groups are in the same orientation in all
the serine proteases. Their roles are basically as follows: the imidazole (His) acts as a general
base-general acid, first to activate the serine OH for nucleophilic catalysis, then the leaving
group (by general acid cat.), then as a general base it activates water to attack the covalent
acyl-enzyme intermediate. The Asp serves to orient the His side chain and to provide an
appropriate electrostatic environment.
EXAMPLES:
• Trypsin and Chymotrypsin are digestive enzymes in the small intestine
• Subtilisin is a bacterial serine protease that is used in laundry detergents
• TADG-14 is a novel extracellular serine protease that has been identified and cloned from
ovarian carcinoma. It is uniquely expressed in ovarian cancer, both in early stage and overt
carcinomas. It is seldom or not at all expressed in normal adult tissues and has not been
detected in other fetal tissues. It offers the potential as a target for therapeutic intervention
through down-regulation of its protease activity.
• NS3/4A is a serine protease in Hepatitis C (HCV) that is important in viral maturation.
• Factor VIIa, Factor Xa, and thrombin are serine proteases in blood coagulation pathway
Trypsin and Chymotrypsin are very well studied serine proteases whose
structures and mechanisms are well understood
They catalyze the hydrolysis of internal peptide bonds (thus an endoprotease). Trypsin
cleaves on the carboxyl side of basic side chains (Lys, Arg), whereas
chymotrypsin cleaves on the carboxyl side of aromatics (Phe, Tyr, Trp)
The active site consists of a “catalytic triad”:
1) Serine, to which the substrate binds
2) Histidine, which has the ability to donate and accept protons.
3) Aspartate, which has the ability to accept protons.
These residues are polar (hydrophilic) so would not ordinarily be
found on the "interior” of a protein.
Though they are in close proximity in the 3D structure, they are not adjacent in the
primary sequence (Ser-195, His-57, Asp-102).
102
First stage in peptide bond hydrolysis: acylation. Hydrolysis of the peptide bond starts with an
attack by the oxygen atom of the Ser195 hydroxyl group on the carbonyl carbon atom of the
susceptible bond. The carbon-oxygen bond of this carbonyl group becomes a single bond, and
the oxygen atom acquires a net negative charge. The four atoms now bonded to the carbonyl
carbon are arranged as a tetrahedron. Transfer of a proton from Ser195 to His57 is facilitated
by Asp102 which (i) precisely orients the imidazole ring of His57 and (ii) partly neutralizes the
positive charge that develops on His57 during the transition state. The proton held by the
protonated form of His57 is then donated to the nitrogen atom of the peptide bond that is
cleaved. At this stage, the amine component is hydrogen bonded to His57, and the acid
component of the substrate is esterified to Ser195. The amine component diffuses away.
Oxyanion
hole
Second stage in peptide hydrolysis: deacylation. The acyl-enzyme intermediate
is hydrolyzed by water. Deacylation is essentially the reverse of acylation with
water playing the role as the attacking nucleophile, similar to Ser195 in the first
step. First, a proton is drawn away from water. The resulting OH- attacks the
carbonyl carbon of the acyl group that is attached to Ser195. As in acylation, a
transient tetrahedral intermediate is formed. His57 then donates a proton to the
oxygen atom of Ser195, which then releases the acid component of the substrate,
completing the reaction.
Oxyanion
hole
(From “Mark’s Basic Medical Biochemistry – A clinical approach”)
Activation Strategies for three classes of Proteases
From: Berg, Tymoczko and Stryer: Biochemistry, 5th edition
How is specificity obtained among trypsin-like serine proteases?
Are all serine proteases evolutionarily related? (NO!)
Catalytic triad: Ser 195, His 57, Asp 102
Catalytic triad: Ser 221, His 64, Asp 32
Which of the following peptides would trypsin cleave?
A. Asp-Leu-Trp-Ala-Leu-Met-Tyr
B. Asn-Thr-Ser-Asp-Ala-Leu-Gly
C. His-Ala-Asp-Val-Asn-Gln-Ala
D. Val-Ala-Val-Lys-Ser-Gly-Phe
E. Phe-Ala-Trp-Ser-Ile-Ala-Gly
The physiological importance of many proteases has made
them an extremely active objects of current research:
•Highly selective small organic compounds are being
investigated as therapeutic leads against many proteases.
• This is one of the hottest areas of drug development in the
biotech and pharmaceutical industry
• Many compounds are in preclinical or clinical trials.
• Many of these compounds will likely be drugs by the time
you are doctors!
Summary Points on Enzyme Catalysis:
1) Understand what is meant by transition state stabilization.
2) Know which amino acid residues can be involved in acid-base catalysis.
3) Understand that covalent catalysis results from the transient formation of a covalent bond
involving a nucleophile and an electrophile.
4) Recognize the general classes of enzymes, including oxidoreductases, transferases,
hydrolases, lyases, isomerases and ligases.
5) Recognize the physiological importance of Proteases.
6) Understand that proteases use nucleophilic attack by either covalent catalysis or
activation of a water molecule, along with acid-base catalysis.
7) Know what is meant by a serine protease.
8) Have a general understanding of covalent catalysis by Trypsin.
9) Understand that the specificity pocket allows different members of the trypsin family of
proteases to use the same catalytic mechanism for proteolysis but with very different
sequence specificities.