Download Enzyme Specificity and Selectivity

Enzyme Specificity and
Selectivity
Introductory article
Article Contents
. Substrate Specificity
Lizbeth Hedstrom, Brandeis University, Waltham, Massachusetts, USA
. Stereospecificity
. Reaction Specificity
Specificity distinguishes enzymes from ordinary chemical catalysts. Specificity is evident in
both discrimination between substrates and control of reaction outcome. Specificity arises
from the three-dimensional structure of the enzyme active site.
Substrate Specificity
Enzymes have two extraordinary properties: (1) they are
very efficient catalysts, accelerating reactions by as much as
1017-fold while operating in water, at neutral pH and
ambient temperatures; (2) they are exquisitely selective,
being capable of discriminating between closely related
substrates and controlling reactions to yield a single
product. While it is often convenient to consider these
two properties separately, it is important to realize that
they are inextricably intertwined: specificity is expressed in
the rate at which a substrate is transformed to product.
Enzyme specificity is measured by the value of
kcat/Km
Three Michaelis–Menten parameters describe a typical
enzyme reaction: kcat, Km and kcat/Km. The kcat value is the
turnover number; it measures the amount of product
formed per enzyme molecule when all of the enzyme has
bound substrate. It is a complicated kinetic constant that
includes the rate constants for all the steps of the reaction
after substrate binding. For example, kcat 5 k2k3/(k2 1 k3)
for the relatively simple reaction of eqn [I]. Since some
selection may occur during substrate binding, kcat is an
inadequate measure of substrate specificity. Km is also a
complex kinetic constant; it is an apparent dissociation
constant of all enzyme-bound substrate complexes. For
eqn [I], Km 5 k3(k 2 1 1 k2)/[k1(k2 1 k3)]. Like kcat, Km is
also an inadequate measure of specificity: it does not take
into account the rate of substrate turnover. In contrast,
kcat/Km provides an accurate measure of specificity. This
parameter is an apparent second-order rate constant for
the reaction of free enzyme and free substrate. Since
different substrates will compete for free enzyme, a
comparison of the values of kcat/Km describes specificity.
Not surprisingly, kcat/Km is also known as the specificity
constant. Note that for eqn [I], kcat/Km 5 k1k2/(k 2 1 1 k2).
This expression includes all of the steps up to and including
the first irreversible step, k2 in this case. These are the only
steps that determine specificity – once a substrate
. Summary
completes the first irreversible step, it is committed to form
product and no further discrimination can occur.
k9
k:
k<
7 ‡ 8 )* 78 ! 7; ! 7 ‡ ;
k
‰=Š
9
The reactions of serine proteases illustrate
enzyme specificity
The specificity of enzyme catalysis is best appreciated in
contrast with chemical catalysis. For example, when
peptide hydrolysis is catalysed by acid, every peptide bond
is hydrolysed and the products are single amino acids
(Figure 1a). In contrast, serine proteases hydrolyse peptides
at discrete sites. For example, trypsin hydrolyses peptide
bonds adjacent to positively charged residues such as lysine
and arginine. The value of kcat/Km for hydrolysis at
positively charged residues is approximately 104 times
greater than that for hydrolysis at other residues. Moreover, while l- and d-peptide bonds are hydrolysed with
equal efficiency in acid, only peptide bonds comprising
l-amino acids are hydrolysed by trypsin. Similarly,
chymotrypsin hydrolyses peptide bonds adjacent to large
hydrophobic residues such as tryptophan, phenylalanine
and tyrosine, while elastase hydrolyses peptides at small
aliphatic residues such as alanine and valine. Thus, the
products of enzyme-catalysed peptide hydrolysis will vary
greatly depending upon the enzyme utilized.
The serine protease reaction has three steps, as shown in
eqn [II]: formation of a noncovalent enzyme–substrate
complex; formation of an acyl-enzyme intermediate with
the active site serine (acylation); and hydrolysis of the acylenzyme intermediate (deacylation). (Note that eqn [II] is
formally the identical to eqn [I]. Therefore the expressions
for kcat, Km and kcat/Km in terms of individual rate
constants are the same as described above.) The individual
rate constants for these steps can be derived from steadystate kinetics or determined directly in pre-steady-state
experiments. Comparison of the reactions of trypsin with
substrates containing either lysine or phenylalanine
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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Enzyme Specificity and Selectivity
Ala + Arg + Glu + 2Gly + Lys + Leu + Phe + Pro + 2Ser + Thr + 2Val
+
H /H2O
Val-Leu-Gly-Ser-Lys-Ser-Phe-Val-Pro-Gly-Thr-Arg-Ala-Glu
trypsin
Val-Leu-Gly-Ser-Lys + Ser-Phe-Val-Pro-Gly-Thr-Arg + Ala-Glu
(a)
L -Peptide
Transition state
−
+
NH 3
N
H
R
H
N
C
NH
O
R
H
O
C
NH
H
N
O
NH
R′
CO2
+
NH 3
+
N
H
O
R
C
NH
H
O
R′ NH 2
CH 2
CH 2
CH 2
CH 2
H
−
C
NH
CO2
NH 3
N
H
CH 2
CH 2
CH 2
CH 2
O
−
−
CO2
H
C N
NH
−
O
NH
O
NH
CH 2
CH 2
CH 2
CH 2
H
C
RCO
E+
NH Lys CO2
O
R′
D-Peptide
−
CO2
+
NH 3
N
H
CH 2
CH 2
CH 2
CH 2
O
R′
H
N C
NH
H
NH
NH
No reaction
C
O
H
O
R
(b)
Figure 1 The hydrolysis of peptide bonds. (a) Comparison of the acid-catalysed and trypsin-catalysed reactions. (b) The active site of trypsin.
residues at the site of hydrolysis reveals that most of the
substrate discrimination occurs in the acylation step.
Trypsin does bind lysine-containing substrates with
10-fold higher affinity than that for phenylalaninecontaining substrates; this difference in binding affinity is
not sufficient to account for the 104-fold difference in kcat/
Km. However, once bound, lysine-containing substrates
2
react 103-fold faster than the phenylalanine-containing
substrates. Thus, enzyme specificity is quantitatively
derived from discrimination in the rate of chemical
transformation, not from discrimination in substrate
binding.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Enzyme Specificity and Selectivity
O
k1
E + RCONHR′
E•RCONHR′
k2
E
O
C
R
k −1
OH
OH
R′NH2
k3
HOH
E + RCO2H
[II]
OH
Enzyme–substrate interactions approximate
those of a lock and a key
The specificity of trypsin, chymotrypsin and elastase arises
from the three-dimensional structure of their respective
active sites. Although the overall structures of these
proteases are very similar, each enzyme has an active site
that is sterically and electrostatically complementary to its
substrate. Both trypsin and chymotrypsin have a deep
pocket at their active sites; the trypsin pocket contains a
negative charge that can complement the positive charge of
the substrate, while the chymotrypsin pocket is hydrophobic, thus accounting for the preference for hydrophobic substrates (Figure 1b). Elastase has a shallow pocket
that can only accommodate small residues. The pockets are
arranged such that the carbonyl group of an l-amino acid
residue is positioned to interact with the catalytic serine,
the NH group makes a hydrogen bond to a main-chain
carbonyl on the enzyme, and the a-hydrogen fits snugly in
the active site (Figure 1b). Thus, substrate specificity is
determined by the accumulation of noncovalent forces:
hydrogen bonding, steric, electrostatic, van der Waals and
hydrophobic. These interactions are reminiscent of the
relationship between a lock and key: hydrophobic parts of
the substrate bind in hydrophobic pockets on the enzyme,
negative charges of the substrate interact with positive
charges on the enzyme, and so forth. The active-site
structure can also account for the stereospecificity of the
serine protease reactions (Figure 1b). A d-peptide can bind
at the active site, with the side-chain occupying the pocket
as in an l-peptide. The a-hydrogen must also occupy the
same position as in the l-peptide: the other groups are too
bulky to occupy this space. Therefore, the carbonyl group
of a d-peptide will not be positioned adjacent to the
catalytic serine, and the enzyme cannot catalyse the
hydrolysis of the d-peptide bond.
Serine proteases are good examples of how steric and/or
electrostatic exclusion can determine substrate specificity:
a substrate that is too big, or of the wrong charge, cannot
enter the active site. Enzymes can also discriminate against
smaller substrates even though they are not excluded from
the active site. For example, chymotrypsin hydrolyses
peptide bonds adjacent to phenylalanine residues much
more rapidly than those adjacent to alanine residues,
although the smaller side-chain of alanine is clearly not
prevented from entering the active site. The lock and key
analogy can also be used to understand such discrimination against smaller substrates. A lock is exactly complementary to its key; this alignment allows the key to move
the tumblers inside the lock and engage the locking
mechanism. In contrast, while a smaller key may fit inside
the lock, it will not move the tumblers. Likewise, a
phenylalanine side-chain fits snugly in the chymotrypsin
binding pocket. This interaction aligns the peptide bond in
the optimal orientation with the catalytic residues of the
enzyme and the reaction will be rapid. Although the
alanine side-chain can also enter the binding pocket, it will
not be firmly bound like the phenylalanine side chain;
therefore, the binding energy of the alanine side-chain is
insufficient to precisely align the peptide bond, and the
reaction will be slower.
Enzyme active sites are complementary to the
transition state of the reaction
While the lock and key analogy is useful for understanding
enzyme–substrate interactions, it is important to remember that an enzyme active site is not simply complementary
to the substrate. Such an enzyme would merely stabilize the
ground state of the substrate, not accelerate the reaction.
Catalysis results from selective stabilization of the transition state. Therefore, the enzyme active site must be
complementary to the transition state of the reaction. This
complementarity to the transition state produces a
corresponding destabilization of the ground state. As
stated by J. B. S. Haldane, ‘The key does not fit the lock
quite perfectly, but exercises a certain strain on it.’
In the serine protease example, the transition state will
have a tetrahedral structure with negative charge developing on the carbonyl carbon (Figure 1b). This tetrahedral
oxyanion is stabilized by hydrogen bonds to two mainchain amide NH groups. However, these same hydrogen
bonds to the substrate activate the carbonyl, priming it for
reaction. Thus these hydrogen bonds strain the substrate;
this strain is compensated in the favourable interactions
between the negatively charged pocket of trypsin and the
positively charged side-chain of the substrate. This is an
example of a phenomenon William Jencks has described as
the Circe effect: favourable interactions with one part of
the substrate pay for unfavourable interactions (i.e.
destabilization of the substrate) at the site of chemical
transformation. The favourable interactions are used to
‘lure’ the substrate into the active site; the destabilization
promotes the chemical transformation. Such destabilization includes desolvation and conformational restriction
as well as electrostatic interactions. For example, the
energetic cost of removing a substrate from water is very
high; therefore, the affinity of the substrate for the enzyme
is weak. However, chemical reactions proceed much faster
in the absence of water. Thus, the cost of desolvating the
substrate earns a large payback in the acceleration of the
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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Enzyme Specificity and Selectivity
reaction. The Circe effect explains why specificity is not
simply determined by the affinity of substrate binding: the
favourable enzyme–substrate interactions are expressed in
reaction rate, not in substrate affinity.
Enzymes can change conformation in
response to their substrate
The lock and key analogy falls short in another regard.
Whereas locks and keys have rigid structures, both
enzymes and substrates are flexible. Substrate conformation can adapt to fit the enzyme active site. For example,
while a peptide may not be conformationally constrained
in solution, it must assume a fixed, extended conformation
when bound to trypsin. Likewise, enzyme conformation
can change in response to substrate binding. Daniel
Koshland first proposed this ‘induced fit’ hypothesis: the
binding of substrate can convert enzyme from an inactive
conformation into an active one, by orienting catalytic
residues, structuring a binding site for a second substrate,
or closing the active site to exclude water. The adaptation
of the active site to the substrate provides another
mechanism of substrate discrimination.
Hexokinase provides a classic example of induced fit.
This enzyme catalyses the transfer of the g-phosphate
group from ATP to the 6-hydroxyl group of glucose. The
reaction with glucose is 107-fold more favourable than that
with water as measured by kcat/Km. ATP binds poorly to
hexokinase in the absence of glucose. The g-phosphate of
ATP is disordered in this complex; this disorder suppresses
the reaction with water. When glucose binds, hexokinase
assumes a more compact structure; glucose is buried inside
the enzyme with only the 6-hydroxyl exposed. This
desolvation will promote catalysis, and the conformational
change also increases the affinity of ATP. In addition, this
conformation activates ATP, presumably fixing the position of the g-phosphate. The activation of ATP is apparent
when glucose is replaced with a nonreactive analogue such
as lyxose. Since lyxose cannot react with ATP, the reaction
with water is accelerated by default: the value of kcat/Km for
the hydrolysis of ATP increases 800-fold. This is an
example of substrate synergism: the binding of one
substrate to the enzyme triggers a conformational change
that activates the second substrate.
Some enzymes utilize proofreading reactions
to increase specificity
A stringent requirement for substrate selection is found in
DNA replication, where the incorporation of the wrong
nucleotide can have catastrophic effects on cell replication.
DNA polymerase makes errors at the astoundingly low
frequency of one error for every 108 –1012 nucleotides.
However, if substrate discrimination were based on
Watson–Crick base-pairing interactions alone, DNA
4
polymerase would make errors at a rate of one in every
104 nucleotides. The high fidelity of DNA polymerase is
achieved by two levels of substrate selection. First, a
nucleotide triphosphate is incorporated into the growing
DNA chain. In the event that a mismatched nucleotide is
inadvertently added, an editing reaction occurs at a second
active site that removes the mismatched nucleotide. The
error frequency for the overall reaction is the product of the
frequencies of each separate active site; that is, if each site
makes one error in 104 nucleotides, the overall reaction will
have one error in 108 nucleotides. A comparable editing
mechanism is observed in the enzymes that attach amino
acids to their cognate tRNAs.
Stereospecificity
Stereospecificity also distinguishes enzyme reactions from
ordinary chemical catalysis. The stereospecificity of
enzymes is most dramatically illustrated by the reaction
of dehydrogenases. As first demonstrated by Frank
Westheimer, these enzymes discriminate between the
hydrogens at C4 of NADH even though C4 is a
symmetrical centre. These hydrogens are prochiral: the
replacement of HR with a deuterium will produce an R
chiral centre, while the replacement of HS with a deuterium
will produce an S chiral centre. Vernon Anderson has
shown that HR is transferred in greater than 99.999 998%
of the turnovers of lactate dehydrogenase (Figure 2). As in
the case of the serine proteases, the stereospecificity of the
lactate dehydrogenase can be rationalized from the threedimensional structure of the enzyme active site and the
relative positions of the pyruvate and NADH binding sites
(Figure 2): pyruvate is bound by interactions at its carboxyl
and carbonyl oxygens, while the nicotinamide ring is
oriented by the interactions of its carboxamide group.
These interactions place HR adjacent to the carbonyl
carbon of pyruvate, ideally positioned for transfer. In
contrast, HS points away from pyruvate; clearly the
transfer of HR will be preferred over HS. However, these
interactions must prevent the occasional rotation of the
nicotinamide, with subsequent transfer of HS. The
energetic barrier for this rotation must be approximately
42 kJ mol 2 1 to account for the magnitude of the preference
for HR. The structural basis of this discrimination remains
a mystery.
Reaction Specificity
Chemical reactions rarely proceed with 100% yield of a
single product: high-energy intermediates usually decompose via several pathways to generate several different
products. In contrast, the high-energy intermediates in
enzyme reactions routinely follow a single pathway to
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Enzyme Specificity and Selectivity
OH
H2 N
Nicotinamide
R
O
HS
N
(C4)
HR
H2 N
O
C
C
O H2 N
C
Pyruvate
O
CH3
HN
NH
H2 N
N
H
NH2
C
NH
OH
H2 N
O
R
HS
N
H2 N
O
HR
C
O
H2 N
C
N
H
C
HO
HN
N
CH3
H2 N
C
NH2
NH
Figure 2 The lactate dehydrogenase reaction.
achieve 100% yields of a single product. This phenomenon
is illustrated by the reaction of triose-phosphate isomerase.
This enzyme catalyses the interconversion of glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate
(DHAP) via an enediol intermediate (Figure 3a). However,
in solution the enediol intermediate is not protonated to
form DHAP. Instead, the enediol decomposes to methylglyoxal via the elimination of phosphate. Methylglyoxal is
not observed in the enzyme-catalysed reaction. The
elimination reaction requires that phosphate assume a
position orthogonal to the plane of the enediol. In this
position, the C–O bond will have the maximum overlap
with the orbitals of the double bond. The enzyme prevents
the elimination reaction simply by holding the phosphate
in the plane of the enediol. Thus, triose-phosphate
isomerase controls the reaction of the enediol intermediate
by constraining its conformation. The reaction of triosephosphate isomerase is an example of stereoelectronic
control.
The ability of enzymes to harness the versatility of
pyridoxal phosphate and similar cofactors is another
example of stereoelectronic control. Pyridoxal phosphate
is required in a diverse set of reactions involving amino
acids: racemization, decarboxylation, transamination, belimination/replacement and g-elimination/replacement
(Figure 4a). Pyridoxal phosphate chemistry begins with
the formation of an imine with the amino group of an
amino acid (Figure 4b). The next step is generation of a
carbanion at the a-carbon of the amino acid. This
carbanion can be formed by extraction of the a-proton
or by decarboxylation of the amino acid. The pathway for
formation of the carbanion is controlled by the orientation
of the substrate – as in the case of triose-phosphate
isomerase, the departing group must be positioned
orthogonal to the plane of the imine double bond. This
carbanion will undergo further transformations, including
protonation, deamination and elimination reactions. In
solution, all of these reactions can occur, producing a
mixture of products. However, the enzyme reactions are
tightly controlled by the orientation of the substrate on the
enzyme and by the positioning of acidic and basic residues
in the enzyme active site. Thus, despite the reactivity of the
pyridoxal phosphate cofactor, each enzyme catalyses a
single chemical transformation.
Perhaps the most striking example of stereoelectronic
control is found in the biosynthesis of sesquiterpenes.
These compounds contain 15 carbons that can be arranged
in over 200 different carbon skeletons, some of which are
shown in Figure 5. This varied array derives from a single
precursor, farnesyl diphosphate, via the rearrangement of
an allylic carbocation intermediate. While in solution such
carbocation rearrangements produce a hopelessly complex
mixture of compounds, the enzyme-catalysed rearrangements produce a single compound. The enzymatic
rearrangement reactions are presumably controlled by
the conformation of farnesyl diphosphate on the enzyme
and the electrostatic surface of the enzyme active site.
Summary
Specificity is a hallmark of enzyme catalysis; it is
inseparable from catalytic efficiency, the other hallmark
of enzyme reactions. Specificity arises from the threedimensional structure of the enzyme active site; this site is
complementary to the transition state of the reaction. The
substrate fits snugly within the enzyme active site,
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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Enzyme Specificity and Selectivity
O G3P
H
C
C
H
H
OH
C
OH Enediol
C
2_
H
CH 2OPO3
OH DHAP
C
H
O
C
H
2_
CH 2OPO3
OH
C
2_
OPO3
H
Rotates in solution
H
C
2_
O3PO
H
H
OH
C
Methyl glyoxal
O
O
OH
C
OH
C
H
C
C
C
H
H
Pi
C O
CH 3
H
Figure 3 The triose-phosphate isomerase reaction.
R
H
–
Racemization
H
C
CO 2
R
C
+
–
CO 2
+
NH 3
NH 3
CO 2
R
Decarboxylation
H
R
C
CO 2–
H
C
+
NH 3
+
–
H
–
RCOCO2 RCH(NH 3 )CO 2
R
Transamination
H
+
NH 3
O
–
CO 2
C
+
NH 3
R
C
–
CO 2
X
β-Elimination/replacement
H
CH2X
–
CO 2
C
+Y
H
CH 2Y
–
CO 2
C
+
+
NH 3
NH 3
X
γ-Elimination/replacement
H
CH 2CH 2X
CO 2–
C
+
NH 3
+Y
H
CH 2CH 2Y
–
CO 2
C
+
NH 3
(a)
Figure 4 Pyridoxal phosphate chemistry. (a) Reactions of pyridoxal phosphate. (b) Stereoelectronic control of carbanion formation in pyridoxal
phosphate reactions.
6
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Enzyme Specificity and Selectivity
PPi
Farnesyl diphosphate
⊕
OPPi
Pentalenene synthase
Trichodiene synthase
H
Bisabolene synthase
Bergamotene synthase
Aristolochene synthase
and so on
Figure 5 The biosynthesis of sesquiterpenes.
optimally aligned to react with the catalytic residues. In
addition, the conformation of the substrate is constrained,
which will control the course of the reaction.
Further Reading
Cane DE (1990) Enzymatic formation of sesquiterpenes. Chemical
Reviews 90: 1089–1103.
Fersht AR (1985) Enzyme Structure and Mechanism, 2nd edn. New
York: WH Freeman.
Hedstrom L (1996) Trypsin: a case study in the structural determinants
of enzyme specificity. Biological Chemistry 377: 465–470.
Jencks WP (1980) Binding energy, specificity, and enzymatic catalysis:
the Circe effect. Advances in Enzymology and Related Areas of
Molecular Biology 43: 219–410.
Knowles JR (1991) To build an enzyme.... Philosophical Transactions of
the Royal Society London Series B 332: 115–121.
Koshland DE (1958) Application of a theory of enzyme specificity to
protein synthesis. Proceedings of the National Academy of Sciences of
the USA 44: 98–104.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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