Binding and Catalysis

Michael D Toney, University of California Davis, California, USA

Binding and catalysis in the biological context are defined as the formation of a relatively long-lived and specific complex between a macromolecular catalyst and a substrate molecule, which subsequently undergoes a chemical transformation much faster than would occur in the absence of the macromolecular catalyst.

Secondary article
Article Contents
. Introduction . Entropy Loss . Binding and Reaction Specificity . Ground State Destabilization . Transition State Stabilization . Summary

Introduction
The formation of stable complexes in catalytic reactions is not unique to biological molecules. Zeolites and palladium-on-carbon, for example, both bind one or more reactants and activate them for catalysis in the complex thus formed. Catalysts such as these commonly show little substrate specicity, regioselectivity, or stereospecicity. Enzymes, on the other hand, commonly bind only one reactant (two or more if required for the reaction) and allow a single stereospecic chemical transformation to occur. They are unique in that the forces of evolution tailor them to the specic metabolic needs of the organism. The plasticity of their protein or nucleic acid structures (in the case of RNA-based and DNA-based catalysts) and their tolerance of structural variation additionally allow enzymes to employ catalytic mechanisms not available to nonproteinaceous catalysts, namely those that involve the use of binding energy for catalytic purposes. Substrate binding in enzyme-catalysed reactions is a physical process that is distinct from the chemical step(s) interconverting substrate and product. The chemical events (i.e. bond breaking and/or making) occur in the conversion of the enzymesubstrate complex to the enzymeproduct complex via a transition state. This is a unimolecular event. The physical binding step pays the approximately 20 kJ mol 2 1 entropic cost of bringing together two reacting moieties (enzyme and substrate) into a single complex. The currency in this transaction is a balance of two eects. The rst is favourable enthalpic interactions made between the enzyme and substrate in the complex. The second is the potential increase in entropy of water molecules that may have been ordered by the substrate in solution or by the active-site pocket. Enzyme-catalysed reactions frequently involve more than one substrate. In these cases, energy available from binding interactions is utilized to form the trimolecular complex of enzyme and the two substrates, when the reaction occurs by a sequential kinetic mechanism. This binding reduces the molecularity of the chemical step(s) to one, thus gaining minimally the factor of approximately 3000 described above for each decrement in molecularity. Orientational factors also come into play in sequential multisubstrate reactions. That is, the details of the geometry of juxtaposition in the active site can be important, and an enzyme can control these. An extreme but illustrative example of this eect is the hypothetical case of a protein that binds two substrates such that they are separated from one another by a distance prohibitive for reaction. This protein would act not as a catalyst of the reaction, but as an inhibitor; that is, the presence of the protein would suppress the nonenzymatic reaction. An enzyme therefore needs to bind two substrates such that the reacting groups are appropriately disposed geometrically. The stringency for what constitutes appropriate disposition can vary between reactions. Phosphoryl transfer and carbonyl addition reactions, for example, will have dierent requirements. Enzymes use binding energy to enforce these reaction-specic orientational requirements. Comparisons of series of homologous intramolecular versus intermolecular reactions of small molecules have historically been used to deconvolute the contributions of
1

Entropy Loss
Entropy generally decreases on going from reactants to the transition state in chemical reactions. This makes a positive (unfavourable) contribution to the activation free energy, since DG{ 5 DH{ 2 T DS{. A bimolecular reaction involves two molecules coming together to form a single species in the transition state. Thus, the translational and rotational entropy of one molecule must be lost simply in bringing the reactants into a productive collision. One might expect this entropy loss to be largely independent of the nature of the reactants. Bruice and Benkovic (1965) have shown that, for a diverse group of approximately 40 reactions in solution, when one divides the measured T DS{ for a reaction by the kinetic order, a value of 20 kJ mol 2 1 is obtained. This value is thus an experimental average of the cost of losing one set of translational and rotational degrees of freedom on going from ground to transition state in solution. This energy translates into a kinetic factor of approximately 3000. That is, if a reaction is simply converted from a bimolecular to a unimolecular reaction, all other factors being equal, the unimolecular reaction will proceed approximately 3000-fold faster.

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Binding and Catalysis

entropy to reaction barriers. Bruices group revisited this subject computationally (Lightstone and Bruice, 1997). For a series of anhydride formation reactions in which the carboxylate and carbonyl groups are progressively held more closely apposed they nd that the entropy of activation does not vary signicantly. Instead, the increases in reaction rate are realized via decreases in the enthalpies of activation. This occurs by a ground state destabilization mechanism (below) in which the electrostatic cost of bringing together the reacting moieties is paid in the synthesis of the constrained molecules. This illustrates the potential for ground state destabilization mechanisms in enzyme catalysis.

Binding and Reaction Specificity

The question of how an enzyme determines binding specicity for one molecule is fundamentally important to enzyme catalysis. Triose-phosphate isomerase is a prototypical enzyme in many ways. It catalyses the reversible interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The enzyme has several places on both the substrate and the product where it can make binding interactions. A total of approximately eight hydrogen bonds with the oxygen atoms of each substrate are, in principle, possible. Assuming that two of these are ion pairs with the phosphate group, one can calculate from average experimental hydrogen bond energies that the total binding energy could be 2 58.5 to 2 75 kJ mol 2 1. For a standard state of 1 mmol L 2 1 substrate, this would correspond to a dissociation constant, Kd, of approximately 10 2 12 mol L 2 1. Given that the in vivo concentrations of these substrates are in the millimolar range, this low value of Kd is clearly unnecessary. The measured Km values for this enzyme are approximately 1 mmol L 2 1. It would probably be counterproductive to have such tight binding since low Kd values generally translate into small dissociation rate constants. This can potentially lead to product dissociation limiting the rate of the overall reaction. An alternative one might imagine is to bind only the phosphate group tightly, but this has the disadvantage that other small phosphate esters might also bind tightly because they contain the only recognition element used by the enzyme. Thus, it appears that the best means for enzymes to recognize specically one substrate is to make several weak interactions over the entire molecule. This has the eect of sterically restricting the size and shape of the binding pocket, as well as its electrostatic topology, to that of the cognate substrate. A distinct mechanism by which enzymes can restrict their substrate specicity is that of induced t, rst propounded by Koshland (1958). Enzymes have been observed crystallographically to undergo large-scale con2

formational changes on binding substrate(s). Although this was unknown at the time, Koshland proposed that conformational changes on binding substrate could be used to discriminate between two substrates. A clear example of this mechanism is the reaction catalysed by hexokinase, the phosphorylation of glucose by ATP. The intrinsic reactivity of the hydroxyl group of the sugar is not substantially dierent from that of water, which is everpresent in biological systems at a concentration of 55 mol L 2 1. The enzyme uses a conformational change to surround the substrate. In doing so, it positions itself to utilize potential binding interactions with remote parts of the sugar as a source of energy for reducing the barrier to the chemical step. Such utilization of binding interactions in catalysis are not possible for the small water molecule. An enzymesubstrate complex may have more than one potential reaction pathway available to it, but the usual experimental observation is that a single reaction product is produced. Thus, the question of how an enzyme, through binding, directs the reaction of the complex through a unique pathway is pertinent to this discussion. One of the conceptually simplest mechanisms by which enzymes can control reaction specicity through binding is via stereoelectronic eects. These commonly occur in reactions where developing charge is stabilized through resonance interactions. The formation of a carbanion, for example, generates a p orbital with a pair of electrons. The p orbital has a well-known symmetric bimodal structure. The degree to which this orbital interacts with adjacent p orbitals of a conjugated p system is dependent on the angle between the p orbital containing the unshared electrons and the orbitals of the p system. The interaction is greatest when the orbitals are aligned parallel, and least when they are orthogonal. The interaction energy varies with the cos2 of the angle between the orbitals. Stereoelectronic eects are well documented in organic chemistry, but have received less attention in enzymology. The concept of stereoelectronic eects has had a strong inuence in research on pyridoxal phosphate-dependent enzymes. Dunathan (1966) proposed, on the basis of thenrecent ndings of organic chemists, that these enzymes could enforce reaction specicity by maintaining a certain orientation, depending on the reaction to be catalysed, about the Ca N bond in the common aldimine intermediate. Figure 1 shows an aldimine intermediate formed between an amino acid and pyridoxal phosphate by addition of the substrate a-amino group to the coenzyme aldehyde function. This is the rst and common step in all pyridoxal phosphate-catalysed reactions (except for glycogen phosphorylase, which uses pyridoxal phosphate noncanonically). In Figure 1, the p orbitals of the conjugated p system constituted by the aldimine bond and the pyridine ring of the coenzyme are shown. The aldimine is orientated about the Ca N bond such that loss of the carboxylate group via decarboxylation will yield a carbanion that is maximally stabilized by resonance

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Binding and Catalysis

2O

3PO

CO2

+HN N+ O H H
Figure 1 Aldimine formed between an a-amino acid and pyridoxal phosphate. The p orbitals of the conjugated aldimine/pyridine ring p system are 2 shown, as is the sp3 orbital of the Ca CO2 bond. The latter is orientated such that maximal stereoelectronic effects will be gained in the transition state for 2 decarboxylation. As the Ca CO2 bond breaks, the nascent p orbital will be maximally aligned with the p orbitals of the aldimine/pyridine ring p system, thereby maximally stabilizing the carbanionic character at Ca. Binding interactions between enzyme active sites and the aldimine maintain the optimal conformation.

H3C

2 interactions with the p system; the Ca CO2 bond is perpendicular to the plane of the coenzyme ring. That is, 2 bond begins to break, the nascent p when the Ca CO2 orbital with its unshared pair of electrons forming on Ca will be maximally stabilized when it is aligned parallel to the p orbitals of the adjacent p system, since this arrangement maximizes orbital overlap. Stereoelectronic control of reaction specicity then originates simply in the enzyme orienting one bond to Ca such that the nascent p orbital that develops from it is aligned with the p orbitals of the p system. This can be achieved by making specic binding interactions between enzyme and the components of the substrate. There are several examples of the importance of stereoelectronic eects in pyridoxal phosphate-dependent enzymes. Possibly the best example is that of the prototypical pyridoxal phosphate-dependent enzyme aspartate aminotransferase, which has been well studied both crystallographically and kinetically. This enzyme catalyses the removal of the a-proton of aspartate in the transamination reaction. The crystal structure of amethylaspartate bound to the enzyme shows that the Ca CH3 bond, structurally analogous to the reactive Ca H bond of aspartate, is orientated perpendicularly to the plane of the coenzyme ring. Stereoelectronic eects play a role in a wide variety of reaction including thiamin diphosphate-dependent decarboxylations, aldose/ketose isomerizations and glycoside hydrolysis. In each case, a specic geometry of the bound substrate, enforced through enzymesubstrate binding interactions, is required to take advantage of this eect.

Ground State Destabilization

The discussion of ground state destabilization and transition state stabilization (below) must be prefaced by stating that these terms refer specically to selective interactions. That is, these are interactions that dierentially aect the ground versus the transition state. If an enzymesubstrate

interaction is made that does not change in energy on going from the ground state to the transition state, then this is a uniform binding interaction and does not aect the rate constant for conversion of the enzymesubstrate complex to the enzymeproduct complex. The discussion below refers only to dierential interactions. There are two basic mechanisms for ground state destabilization: geometric and electrostatic. The concept of geometric distortion of bound substrate away from the ground state towards the transition state structure has a special historical place in enzymology. Lysozyme was the rst enzyme structure to be solved using X-ray crystallography. This enzyme hydrolyses the polysaccharide portion of bacterial cell walls. Phillips and co-workers subsequently proposed, from the structure of a trisaccharide bound to the enzyme, that it distorts the sugar containing the reactive anomeric carbon from the more stable chair conformation towards the less stable sofa conformation (Imoto et al., 1970). This is a step in the direction of the transition state, which is a planar oxocarbonium ion. This proposal stirred great debate and spurred many experiments aimed at testing the hypothesis. Computational work has suggested that protein structures are too exible to enforce the geometric distortion, while experimental studies using various substrate analogues have yielded results consistent with the proposal. Kirby (1987) pointed out that not only would a geometric distortion drive the reaction physically but it would also allow stereoelectronic eects to facilitate the reaction. One cannot object to electrostatic ground state destabilizations on the grounds that enzymes are not rigid enough to enforce them. In fact, several examples exist in the literature where strong experimental evidence supports this eect. The rst step in the reaction catalysed by triosephosphate isomerase, introduced above, in the conversion of dihydroxyacetone phosphate to glyceraldehyde 3phosphate, is deprotonation of C1 to form an enediol intermediate. A histidine residue in the active site, His95, has been shown by infrared spectroscopy and mutagenesis to polarize the C2 carbonyl group in the ground state such
3

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Binding and Catalysis

that formation of the enediol intermediate is facilitated (Komives et al., 1991). Lactate dehydrogenase catalyses the reduction of pyruvate to lactate using NADH as a source of reducing equivalents through a hydride transfer mechanism. In this reaction also, polarization of the carbonyl group will facilitate reaction. A combination of Raman spectroscopy and mutagenesis has been used to demonstrate a clear polarization in the ground state of the carbonyl by His195 and Arg171 (Deng et al., 1994).

Transition State Stabilization

Linus Pauling in 1946 proposed that enzymes work by interacting more strongly with the transition state than with the ground state and this idea shaped enzymologists thinking over the succeeding decades. It is undoubtedly generally correct. Figure 2 dissects the energy changes that occur in an enzymatic reaction using a simplistic diagram of the prevailing current view. The diagram divides the observed free energy prole into two component parts: the selfenergy of the substrate (i.e. electronic energy) and the enzymesubstrate interaction energy (i.e. the binding energy). When substrate binds to the enzyme, the self-

energy of the substrate does not change substantially (in the absence of ground state destabilization). On going from the enzyme-bound ground state to the transition state for the chemical step (where bonds are made and/or broken), the self-energy of the substrate increases, and subsequently decreases on going to the product state. Product dissociation, a physical step like substrate association, does not change the substrate self-energy. The enzymesubstrate interaction energy decreases slightly (favourable substrate association) when substrate binds to the enzyme. A much larger decrease in the enzymesubstrate interaction energy occurs as the substrate goes from the enzyme-bound ground state to the transition state. This is the single most essential component of enzymatic catalysis: the enzyme binds the transition state much more strongly than it does the ground state of the substrate. The enzymesubstrate interaction energy is reduced in magnitude as the product state is reached, and again when product dissociates. The net result, the observed free energy prole, is the sum of the substrate self-energy and enzymesubstrate interaction energy proles. The enzymesubstrate and enzymeproduct complexes both form favourably. The central step, the chemical interconversion of substrate and product, is endergonic since the increase in substrate self-energy is not fully oset by the decrease in energy due

Substrate self-energy Positive Free energy change Observed energy

E+S

ES

+ E S+

EP

E+P

Negative

Enzymesubstrate interaction energy

Figure 2 Schematic of the free energy changes that occur in a simplified enzymatic reaction. The energy levels have been offset from zero for clarity. The substrate self-energy is the electronic energy of the substrate that would be found if it were transferred to the gas phase. The enzyme substrate interaction energy is the binding energy between enzyme and substrate. Formation of either the E-S or E-P complexes occurs with a small decrease (favourable change) in enzyme substrate interaction energy. On going from the E-S or E-P complexes to the transition state for the chemical interconversion, the enzyme substrate interaction energy decreases by a large amount, indicating selective transition state stabilization. The substrate selfenergy does not change on binding to the enzyme (in the absence of ground state destabilization). It undergoes a large increase on going from either the E-S or E-P ground states to the transition state owing to the electronic structure changes that are entailed in the process. The net result is the observed energy profile, which is the sum of the profiles for the substrate self-energy and the enzyme substrate interaction energy. It decreases slightly on substrate binding, and increases in the chemical step since the magnitude of the change in the substrate self-energy is larger than that of the enzyme substrate interaction energy. The barrier in the substrate self-energy is invariant. Thus, selective transition state binding will always increase the catalytic rate constant.

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Binding and Catalysis

to enhanced enzymesubstrate interactions. A simple conclusion follows: the greater the selective transition state binding an enzyme achieves, the greater the rate acceleration, since the changes in substrate self-energy are constant for a given transformation. A spectacular example of selective transition state binding was observed by Fersht and co-workers (Leatherbarrow et al., 1985) with tyrosyl-tRNA synthetase. In the rst step of this reaction, the enzyme catalyses the formation of tyrosyl adenylate from tyrosine and ATP. This is displacement of pyrophosphate by attack of the tyrosine carboxylate on the a-phosphate of ATP. The ground state geometry of the a-phosphate is tetrahedral, which changes to trigonal bipyramidal on going to the direct displacement transition state. This change in geometry is seized upon by the enzyme to generate selective transition state binding interactions. Figure 3 schematically depicts the interactions that occur. The side-chains of
Ground state Thr40 OH O P O O O NH3+ HO O O O P O O HN His45 N O O P O

Thr40 and His45 do not interact with the g-phosphate of ATP in the ground state owing to the tetrahedral geometric constraints of the a-phosphate. As the transition state is approached, the change from tetrahedral to trigonal bipyramidal geometry at the a-phosphate causes a movement of the b- and g-phosphates towards Thr40 and His45 such that hydrogen bonds are formed selectively to the gphosphate in the transition state. In this case, the physical displacement of atoms on going from the ground to the transition state is used to provide selective transition state stabilization in the form of hydrogen bonds, which are strongly dependent on donoracceptor distance. The above example of tyrosyl-tRNA synthetase again illustrates the use of binding energy for catalytic purposes; the binding energy of the g-phosphate group of ATP is more fully realized in the transition state than in the ground state. The use of binding energy for catalytic purposes is a general theme in enzymology. It has been clearly detailed by Jencks (1975), who dubbed it the Circe eect. As discussed above for triose-phosphate isomerase, enzymes generally have substantially more binding interactions available to them than required to attain a physiologically relevant substrate anity. Some of the potential interactions are indeed used to make substrate association slightly favourable under physiological conditions, but the remainder are available for conversion into transition state stabilization and/or ground state destabilization, that is, for catalysis. Many examples of the transduction of binding energy into catalysis exist in the literature.

Adenosine

Summary
The formation of a relatively long-lived enzymesubstrate complex is an essential step in enzyme catalysis. The lowering of kinetic order, as occurs when two substrates combine with an enzyme into a single complex in sequential kinetic mechanisms, results in a rate enhancement of approximately 3000-fold, all other factors being equal. The amount of energy potentially available for enzyme substrate association can be very large. Only a fraction of it is required to provide high specicity for ligand binding, as well as dissociation constants that match the physiological concentrations of substrates. The energy in excess of that required for simple binding is available for catalytic purposes. The transduction of binding energy into catalysis can take many forms. Two basic mechanisms are ground state destabilization and transition state stabilization.

Transition state His45 Thr40 OH O O O O P O O O

Adenosine

N HN O O

O P O

O NH 3+ HO

References
Bruice TC and Benkovic SJ (1965) Bioorganic Mechanisms. New York: Benjamin. Deng H, Zheng J, Clarke A, Holbrook JJ, Callender R and Burgner JW II (1994) Source of catalysis in the lactate dehydrogenase system.

Figure 3 Schematic of ground and transition state interactions made in tyrosyl-tRNA synthetase. The g-phosphate does not interact with Thr40 and His45 in the ground state. The change in the geometry at the aphosphate in the transition state allows the g-phosphate to make hydrogen bonds to Thr40 and His45 selectively in the transition state.

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Binding and Catalysis

Ground-state interactions in the enzymesubstrate complex. Biochemistry 33: 22972305. Dunathan HC (1966) Conformation and reaction specicity in pyridoxal phosphate enzymes. Proceedings of the National Academy of Sciences of the USA 55: 712716. Imoto T, Johnson JN, North ACT, Phillips DC and Rupely JA (1970) Vertebrate lysozymes. In: Boyer P (ed.) The Enzymes, vol. 7. New York: Academic Press. Jencks WP (1975) Binding energy, specicity, and enzymic catalysis: the circe eect. Advances in Enzymology and Related Areas of Molecular Biology 43: 219410. Kirby AJ (1987) Mechanisms and stereoelectronic eects in the lysozyme reaction. CRC Critical Reviews in Biochemistry 22: 283315. Komives EA, Chang LC, Lolis E, Tilton RF, Petsko GA and Knowles JR (1991) Electrophilic catalysis in triosephosphate isomerase: the role of histidine-95. Biochemistry 30: 30113019. Koshland DE Jr (1958) Application of a theory of enzyme specicity to protein synthesis. Proceedings of the National Academy of Sciences of the USA 44: 98104. Leatherbarrow RJ, Fersht AR and Winter G (1985) Transition-state stabilization in the mechanism of tyrosyl-tRNA synthetase revealed

by protein engineering. Proceedings of the National Academy of Sciences of the USA 82: 78407844. Lightstone FC and Bruice TC (1997) Separation of ground and transition state eects in intramolecular and enzymatic reactions. 2. A theoretical study of the formation of transition states in cyclic anhydride formation. Journal of the American Chemical Society 86: 418426.

Further Reading
Jencks WP (1975) Binding energy, specicity, and enzymic catalysis: the circe eect. Advances in Enzymology and Related Areas of Molecular Biology 43: 219410. Jencks WP (1969) Catalysis in Chemistry and Enzymology. New York: McGraw-Hill. Cornish-Bowden A (1995) Fundamentals of Enzyme Kinetics. London: Portland Press. Fersht A (1985) Enzyme Structure and Mechanism. New York: Freeman.

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