Issue Date: February 23, 2004
MUCH ADO ABOUT ENZYME MECHANISMS
What are the underlying forces and mechanisms that enable enzymes to accelerate reactions? "Quite recently, we have found that there are enzymes that enhance reaction rates by factors as large as 1020-fold," says chemistry professor Richard V. Wolfenden of the University of North Carolina, Chapel Hill. "The challenge is to explain how they do this."
Some contend that the issue is all decided--that enzymes generally catalyze reactions by stabilizing transition states (activated complexes), enabling reactants to convert easily to products. But it's frequently a lot more complicated than that.
"It's a fairly heated-up subject right now," says chemistry professor Stephen J. Benkovic of Pennsylvania State University. "We're beginning to understand, after all, that transition-state binding is just a catchphrase. Clearly, that's one way you have catalysis. But now we're getting a better idea of how it actually comes about."
According to transition-state theory, a stable reactant must surmount a free-energy barrier to be converted to a product. This barrier is the transition state, a species whose reacting bonds are midway to being formed or broken, as the case may be. Transition states are "dynamical bottlenecks to reaction, defining the critical point of no return where potentially reacting species convert to products or return to reactants," explains chemistry professor Donald G. Truhlar of the University of Minnesota, Twin Cities. Enzymes can aid reactions by binding to and lowering the energies of transition states, thus making it easier for enzyme substrates to traverse reaction paths.
Most enzymologists believe this is the basic way most enzymes work--that transition-state binding and stabilization are primarily responsible for catalytic rate acceleration. However, this view has been undergoing considerable refinement, as researchers study phenomena that may underlie, accompany, or even supersede transition-state stabilization--such as electrostatics, quantum mechanical tunneling, coupled protein motions, low-barrier hydrogen bonds, and near-attack conformations. But most of these refinements have themselves been subject to some degree of debate or even controversy.
For example, professor of chemistry and biochemistry Arieh Warshel of the University of Southern California, Los Angeles, and coworkers have shown that electrostatic interactions play a predominant role in the energetics of enzymatic reactions. Warshel and coworkers found that electrostatic forces provide most of the energy that enables enzymes to bind transition states more strongly than ground states.
THE PROPOSAL that transition-state stabilization is primarily electrostatic "is gaining wide acceptance," Warshel says. "All enzymes studied up to now by proper simulation methods have been found to work this way."
However, professor of chemistry and biochemistry Thomas C. Bruice of the University of California, Santa Barbara, notes that hydrophobic forces are important, too. Warshel "holds that the only thing that counts is electrostatic interactions," Bruice says, but "the holding of amino acid side chains in position is mainly by hydrophobic forces. To have an enzyme, you obviously need both." Warshel agrees that hydrophobic effects are important in preorganizing the enzyme into a catalytic configuration, "but in the preorganized enzyme," he says, "the effect of the active site environment on the activation barrier is by far dominated by electrostatics."
Other researchers have suggested that catalysis in some enzymes is accompanied by tunneling--a quantum mechanical phenomenon that permits protons or hydride ions to pass through an energy barrier even though they have insufficient energy to do so from a classical (nonquantum mechanical) standpoint. Recent studies on tunneling have demonstrated that classical views of some mechanisms "are a gross oversimplification and cannot adequately explain enzyme catalysis for many systems," says biochemistry professor Nigel S. Scrutton of the University of Leicester, in England.
BASED ON ANOMALIES observed in kinetic isotope effects--the influence of isotopic substitutions on reaction rates--chemistry professor Judith P. Klinman of the University of California, Berkeley, and coworkers first proposed in the 1980s that tunneling plays a key role in certain enzymatic processes, such as hydrogen-transfer reactions. The researchers found tunneling to be a missing component that accounted at least in part for the ability of some enzymes to accelerate reactions much faster than expected when only classical or semiclassical mechanisms were considered.
Simulations carried out by University of Minnesota chemistry professor Jiali Gao, Truhlar, and coworkers have confirmed that tunneling can increase the rate of an enzymatic reaction substantially. "For the proton-transfer reaction catalyzed by methylamine dehydrogenase, we found that tunneling increases the rate constant by a factor of 70, which is equivalent to lowering the free energy of activation by 2.5 kcal per mole" out of a total of about 15 kcal per mole for the catalyzed reaction without tunneling, Truhlar says.
Chemistry professor Sharon Hammes-Schiffer at Penn State and coworkers have investigated hydrogen tunneling in enzyme reactions by a completely different computational approach, and their results agree with those of Gao, Truhlar, and coworkers for two systems: liver alcohol dehydrogenase and dihydrofolate reductase (DHFR). "This agreement provides validation for both methods," Hammes-Schiffer says. In addition, recent experimental data on DHFR tunneling from assistant professor of chemistry Amnon Kohen's lab at the University of Iowa "are in excellent agreement with both theoretical studies," Kohen says.
However, in a review on enzyme theory and simulation [Science, 303, 186 (2004)], Gao, Truhlar, postdoc Mireia Garcia-Viloca, and professor of chemistry and chemical biology Martin Karplus of Harvard University pointed out that tunneling usually has only minor acceleration effects on rates of reactions. "For all systems that have been analyzed in sufficient detail, the contribution is generally relatively small--between a factor of two and 10 at room temperature" compared to the corresponding uncatalyzed reactions, Karplus says. "The maximum contribution is a factor of about 1,000--much smaller than the effects of lowering of the free energy barrier, though still interesting." Lowering the barrier would typically accelerate a reaction by a factor of 106 to 1020, he notes.
Klinman counters that the review treats tunneling as a correction to transition-state theory and that this can be misleading. Tunneling is not necessarily a correction "but an alternate way to look at the catalysis," she says. "There are increasing examples of reactions that show properties that cannot be explained by a tunneling correction and that appear to take place via a full tunneling mechanism."
Warshel emphasizes the importance of comparing enzyme reactions to corresponding uncatalyzed reactions in water, and on that basis, he, like Karplus, rejects the idea that tunneling plays a major accelerating role in reactions. Although tunneling may indeed occur in enzyme reactions, calculations and experiments show that "it does not contribute to enzymatic catalysis since similar tunneling effects exist in the solution reaction," Warshel says. "This was found to be the case even for enzymes with enormous tunneling corrections."
Indeed, chemistry professor Richard G. Finke and coworkers at Colorado State University, Fort Collins, found experimentally, through studies of kinetic isotope effects, that tunneling occurs to an equivalent extent in a hydrogen transfer catalyzed by methylmalonyl-CoA mutase and in the corresponding reaction in solution (C&EN, Sept. 22, 2003, page 29). This suggests that the enzyme was not designed to enhance tunneling, although it's of a type in which tunneling was believed to play an important role.
Nevertheless, regarding the Finke group's study, Klinman believes methylmalonyl-CoA mutase may be an inappropriate enzyme from which to generalize about the use of tunneling by other enzymes. And regarding Warshel's comment about the comparability of tunneling in both solution and enzyme reactions, Klinman replies that this view is "incompatible with most of the available experimental data" on enzymatic reactions in which tunneling occurs and that in many cases the mechanisms of uncatalyzed solution reactions and enzyme reactions aren't strictly comparable.
Another recent development is that "dynamical aspects of catalysis--protein motions--are coming to the fore," Scrutton says, and studies of motions in enzymes "are leading the way in terms of looking at dynamics experimentally."
FOR EXAMPLE, studies of DHFR have revealed that equilibrium motions of coupled networks of mainly conserved residues may help control the enzyme's catalytic mechanism, note Benkovic and Hammes-Schiffer in a recent review [Science, 301, 1196 (2003)]. These motions help move the reaction along by bringing hydride-transfer sites into close proximity, creating a favorable electrostatic environment for charge transfer and lowering the energy barrier for hydride tunneling.
Professors of molecular biology Peter E. Wright and H. Jane Dyson at Scripps Research Institute and their coworkers have used nuclear magnetic resonance spectroscopy (NMR) to determine rates of motions of active-site loops and conformational changes that lead to transition-state stabilization in DHFR. And Gao, Truhlar, and coworkers have found that the xylose isomerase-catalyzed hydride-transfer reaction is strongly coupled to the relative motion of the enzyme's two Mg2+ ions.
The Klinman and Scrutton teams, working independently, have invoked the idea of "gated motion"--active-site deformations that facilitate tunneling. "Both groups have provided quite a bit of experimental data in support of that model, theoreticians have put together a framework that's consistent with it, and computational chemists are simulating reactions to look for evidence of it," Scrutton says. "So there's a nice synergy developing now between the computational chemistry community and the experimentalists in trying to reconcile these different models."
Professor of chemistry and biochemistry Kendall N. Houk of the University of California, Los Angeles, and coworker Xiyun Zhang hope to soon submit a paper describing still another phenomenon they have found to play a major role in many enzyme mechanisms. By assessing the maximum strength of noncovalent protein-ligand interactions, they show that enzymes that accelerate reactions by a factor of more than 1011 (or cofactors of such enzymes) must bond covalently with reacting substrates. "Enzymes change the chemistry from that occurring in water," Houk tells C&EN. "To lower activation barriers, they create new chemistry--new bonds and intermediates that do not form in the corresponding uncatalyzed processes." One researcher comments that the findings are startling and significant, but a couple of others say the paper simply revisits what is already known.
A notion about enzyme mechanisms that generated controversy about a decade ago was the proposal that low-barrier hydrogen bonds (LBHBs) provide an additional way for enzymes to achieve high rates of catalysis. LBHBs are short and strong hydrogen bonds that are shared equally by two bases.
Researchers who have found LBHBs to play key roles in enzyme reactions include biochemistry professors Perry A. Frey and W. Wallace Cleland of the University of Wisconsin, Madison; professor of biochemistry John A. Gerlt of the University of Illinois, Urbana-Champaign; and the late professor of chemistry Paul G. Gassman of the University of Minnesota. For example, Frey points out that physicochemical evidence of various types has confirmed the existence of LBHBs in serine proteases and that the proton of an LBHB in the enzyme subtilisin has been imaged in an ultrahigh-resolution X-ray crystal structure.
However, Warshel rejects the notion that LBHBs are important in catalysis. He and his coworkers have calculated that LBHBs do not stabilize transition states any better than preorganized ionic hydrogen bonds. Chemistry lecturer Adrian J. Mulholland of the University of Bristol, in England, agrees with Warshel. "Experimental and theoretical studies have shown that LBHBs contribute little more than normal hydrogen bonds," he says. Furthermore, "it has been shown for several enzymes in which LBHBs were proposed that they in fact do not exist."
Cleland and Gerlt reply that LBHBs not only exist but can account for at least five orders of magnitude of rate acceleration of certain enzymes, "with other factors providing the rest of the catalysis" [J. Biol. Chem., 273, 25529 (1998)].
MORE RECENTLY, debate has also arisen over a concept by Bruice and Sun Hur that near-attack conformations (NACs)--substrates that adopt reaction-ready shapes--play key roles in accelerating certain reactions, such as that catalyzed by chorismate mutase. According to the researchers, chorismate mutase accelerates its reaction by binding its substrate primarily in its NAC form, rather than by lowering the transition-state barrier per se [J. Am. Chem. Soc., 125, 10540 (2003) and Proc. Natl. Acad. Sci. USA, 100, 12015 (2003)].
Experiments by his group provide "absolute proof that chorismate mutase works by the enzyme creating a proper conformation to go to the transition state," Bruice says. "Then the enzyme just sits back and watches it happen. There's no way around that at all."
He adds that the NAC concept makes it easy to calculate enzyme properties thermodynamically, without recourse to quantum mechanics, if the free energy of reaction has already been determined. "I think the theoretical calculations of Truhlar, Karplus, and so forth are very important, but in certain places this very simple [NAC-based] calculation method is very good."
Warshel, true to form, disagrees with the NAC concept, saying that it's ill defined because it corresponds to an intermediate on the way to the transition state, and intermediate formation is incapable of changing the activation barrier and accelerating a reaction. "The NAC effect is simply the consequence of transition-state stabilization, rather than the reason for catalysis," he says. And Mulholland points out that "the NAC effect is not by itself sufficient to account for catalysis in chorismate mutase. We find that the NAC contribution is 2 to 5 kcal per mole," out of a total of about 10 kcal per mole for the overall reaction. Bruice replies that some of the modeling techniques used by Warshel, Mulholland, and others are limited and can't simulate NACs properly.
Kohen sees some value in the NAC concept. "Bruice is expressing something that many enzymologists feel very strongly about--the notion that the reaction has to go through a geometrical bottleneck and that a substantial role of the enzyme is to bind the reactant into [a reactive] conformation," Kohen says. NACs may not be that significant energetically, but "although the difference in energy between reactant and transition state determines the enzymatic reaction rate, it is of interest to understand the path between them. Bruice's NAC model addresses that path. It looks like the localized restriction of the reactant to a conformation closer to that of the transition state is stabilized by the enzyme."
Cleland also supports the NAC idea. "The concept is very useful for seeing what actually happens in the active site," Cleland says. Problems people have with NACs may be primarily semantic ones, he says.
An advantage of the NAC concept "is that when it works, the calculations are easier," Truhlar concludes. "There is no guarantee that it will work, but there is a considerable amount of new insight to be gained by a deeper look at reaction-ready substrate conformations that resemble transition states."
In any case, controversies over this and other mechanistic proposals show that advances in the area of enzyme mechanisms can be hard to pin down. Sometimes a proposal will seem to make it over the barrier, whereas at other times it may seem to be rolling straight back toward the ground state. Overall, researchers are making considerable progress toward achieving a deeper understanding of the enzymatic process. It's just that progress doesn't always happen at catalytic rates.
Transition-State Theory Has Venerable History
The transition-state theory of enzymes was depicted early on as shown in this figure, which was adapted by University of Munich chemistry professor Georg-Maria Schwab from a paper by Hungarian physical chemist Michael Polanyi [Zeitschrift für Elektrochemie, 27, 143 (1921)] and revised again for C&EN by chemistry professor Richard V. Wolfenden of the University of North Carolina, Chapel Hill.
"Although Polanyi's contribution has not been widely recognized until recently, he put his finger on the single thermodynamic requirement of catalysis, which is universally required of any catalytic system, including enzymes," Wolfenden explains. This is the notion "that any catalyst can enhance the rate of a reaction only to the extent that it binds the substrate more tightly in the transition state than in the ground state."
The figure shows that an enzyme (E) accelerates a reaction in which a substrate (S) is converted to a product (P) because it decreases the free energy difference (vertical distance) between the transition state (ES) and the ground state (ES), relative to the energy difference between the activated substrate (S) and substrate in the corresponding nonenzymatic reaction. By "adsorbed," Polanyi meant what would today be called "bound."
"Polanyi's fundamental idea, reiterated later by his younger contemporaries J. B. S. Haldane and Linus Pauling, led to the development in my laboratory and others of transition-state analogs, stable inhibitors that exploit the special affinity of enzymes for the altered substrate in the transition state," Wolfenden says. "Transition-state analogs have provided much of the driving force for rational drug design during the past 30 years. Michael Polanyi and his student Henry Eyring went on to develop transition-state theory during the 1920s and '30s, and Michael's son John won the Nobel Prize in Chemistry in 1986 for work on reaction dynamics."
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