Protein Science (1998). 7799-802. Cambridge University Press. Printed in the USA. Copyright 0 1998 The Protein Society

RECOLLECTIONS

A golden era for understanding enzyme mechanisms

GORDON G. HAMMES Department of Biochemistry. Duke University Medical Center, Durham, North Carolina 27710

Understanding how enzymes work has fascinated biochemists for many decades. Many different questions can be asked. Why is catalysis so much more efficient than model systems? How is structure related to function? What are the molecular interactions that promote catalysis? How are enzymes regulated? In this Rec- ollection, I will describe my early experiences in research that bear on a small part of enzymology, namely, what are the elementary steps in enzyme catalysis, how can they be studied, and what information does this provide about the general question of how enzymes catalyze chemical reactions so effectively. I apologize in advance for the incomplete referencing, which is in keeping with the idea that these recollections are intended as personal statements rather than extensive reviews.

My original research interests were inclined toward physical chemistry and the dynamics of chemical reactions. When I was a graduate student at the University of Wisconsin (1956-1959). I immediately joined the research group of Bob Alberty, who was carefully dissecting the mechanism of action of fumarase, primar-

Reprint requests to: Gordon G. Hammes, Duke University Medical Cen- ter, 3701, Durham, North Carolina 27710; e-mail: hammeOOl @mc.duke.edu.

Gordon G . Hammes is currently University Distinguished Service Pro- fessor of Biochemistry and Vice Chancellor for Medical Center Academic Affairs at Duke University. Dr. Hammes did his undergraduate work at Princeton University (B.A., 1956). his graduate work in physical chemistry at the University of Wisconsin (Ph.D.. 1959). and postdoctoral work at the Max Planck Institute fur physikalische Chemie in Gottingen, Germany. He joined the faculty of the chemistry department of MIT in 1960 as an instructor, became an associate professor in 1964, and moved to the Chem- istry Department at Cornell University as a professor in 1965. At Cornell, Dr. Hammes was the Horace White Professor of Chemistry and Biochem- istry, served as chair of the Chemistry Department, and was the first di- rector of the Biotechnology Program. He was Professor of Chemistry and Vice Chancellor for Academic Affairs at the University of California, Santa Barbara from 1988-1991 and moved to Duke University in 1991. Dr. Hammes has been on the Executive Committee of the Division of Biolog- ical Chemistry of the American Chemical Society and the Council of the American Society for Biochemistry and Molecular Biology for multiple terms, and was President of the American Society for Biochemistry and Molecular Biology. He has served on numerous editorial boards and as Editor of Biochemistn since 1992. Dr. Hammes has received the Eli Lilly Award in Biological Chemistry (American Chemical Society) and is a member of the American Chemical Society, the American Society for Biochemistry and Molecular Biology, Phi Beta Kappa. Sigma Xi, the Amer- ican Academy of Arts and Sciences, and the National Academy of Sciences.

Dr. Hammes' research interests have centered on biophysical chemistry, including enzyme kinetics and mechanisms. fast reaction techniques, reg- ulation of enzymes, multienzyme complexes, and membrane-bound en- zymes coupled to ion transport. He has published more than 200 scientific articles and several books

Gordon G. Hammes in 1997.

ily by the use of steady-state kinetics. At that point in time, very few enzymes had been carefully studied with steady-state kinetic methods, and the limitations of this approach were not fully ap- preciated. In my view, the most incisive analysis of the steady-state kinetics of enzyme reactions was performed while I was in grad- uate school, by Len Peller, a postdoctoral fellow in the Alberty research group. I still refer graduate students to this paper (Peller & Alberty, 1959). Basically, it was shown that the steady-state rate law is the same, regardless of the number or nature of the inter- mediates, and that the steady-state parameters, Michaelis con- stants, and maximum velocities provide lower bounds to the rate constants of the individual steps. During that same time period, Ed King, a professor of inorganic chemistry, along with Altman, a

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graduate student, devised a schematic method for solving complex steady rate equations. This methodology was later used by Mo Cleland to develop systematically the steady-state rate equations for multi-substrate reactions.

The bottom line of all of these considerations was that steady- state kinetics is unable to give information about the number and nature of reaction intermediates. Steady-state kinetics can provide information about the catalytic efficiency of the enzyme (turnover number), the affinity of the enzyme for the substrates in the steady state (Michaelis constant), lower bounds for the rate constants of individual steps, the stereochemical nature of the active site (by use of different substrates and inhibitors), and the pathways for addition of substrates to the enzyme for multisubstrate reactions. In addition, the pH dependence of the steady-state parameters can provide information about the nature of ionizable groups on the enzyme important for catalysis, as well as limited information about the nature of reaction intermediates. The use of isotopes also can provide useful information about the intermediate steps in the reaction mechanism.

The limitations of steady-state kinetics had been recognized by others, and rapid mixing devices, particularly the stopped-flow, were used to observe directly the intermediates in enzymatic re- actions. The work of Gutfreund and Sturtevant on chymotrypsin and Britton Chance and coworkers on catalase and peroxidase were very significant studies. As an aside, it should be noted that the first enzymes selected for study with transient kinetic methods were selected for their availability in large quantities and stability, rather than for their in.trinsic interest. However, rapid mixing meth- ods have an inherent time limitation, typically a few milliseconds, and were considered difficult experimental tools.

While I was at Wisconsin, Manfred Eigen gave a brilliant sem- inar on relaxation methods, methods for studying reaction rates that extended into the nanosecond time range. Eigen, of course, later received a Nobel Prize for this work, but at that time his work was just becoming known. It is fair to say that he bowled over his audience-I remember Ed King stating that he felt he should be- come a shoe salesman after hearing Eigen’s talk. I immediately decided this was the future for studying enzyme mechanisms and was very fortunate to obtain a National Science Foundation Post- doctoral Fellowship to study with Eigen at the Max Planck Insti- tute in Gottingen, Germany. While I was there, I not only learned to speak German fluently (with a temble accent), and how to build equipment for studying fast reactions, I also had the opportunity to have many stimulating discussions with Eigen and others in the laboratory about kinetics, enzymes, and many other topics. As a side comment, the Eigen laboratory was a fascinating place to work: it had the most sophisticated, up to date electronic equip- ment in the world, and yet did not have a pH meter until the end of my year of study. The pH was determined by using a potenti- ometer to measure the voltage between two electrodes, and the voltage was converted to pH. This was a reflection of the very physical chemical orientation of the laboratory. It is hard to imag- ine a more stimulating intellectual environment.

My first faculty position (1960) was in the Chemistry Depart- ment at MIT, where I proceeded to build a stopped flow, a tem- perature jump, and equipment for measuring ultrasonic absorption, and began applying these methods to the study of enzyme catal- ysis. For the record, my setup “package” was an oscilloscope and the machine shop resources necessary to build a temperature jump. Commercial equipment for studying fast reactions was not readily available, so that knowing how to build complex equipment was

essential for this research. I was provided with one room of ap- proximately 400 square feet, which served as my office and lab- oratory. I was fortunate to have a very talented group of coworkers. Although not all of them will be mentioned explicitly by name, they were responsible for the fortunes of my research program. Especially important to me was the encouragement and support I received from two senior faulty members, Jack Buchanan, who allowed me to have free use of his laboratory and who directed to me my first postdoctoral associate, Renata Cathou, and first bio- chemistry graduate student, Paul Schimmel; and Bert Vallee, who also allowed me to have free use of his laboratory at Harvard. (Renata later became a professor of biochemistry at Tufts Univer- sity School of Medicine, and Paul was a professor of biology at MIT until 1997, when he moved to Scripp’s Institute.)

The first two enzymes we studied were aspartate aminotrans- ferase and ribonuclease. The former was selected because of a very fortuitous meeting with Paolo Fasella at the University of Rome during my postdoctoral year. He was an assistant professor who got stuck with the job of keeping me occupied during my visit. As it turned out, he was married to the sister of a classmate of mine at Princeton and very much wanted to spend some time in the United States. His research was concerned with aspartate amino- transferase, an ideal enzyme for fast reaction techniques because of the many spectral changes known to occur during catalysis and the availability of large amounts of enzyme. Paolo spent a full year at MIT, and our collaboration continued over many years in Boston, Rome, and later, Ithaca. Ribonuclease was selected for study be- cause it is a relatively small enzyme, whose structure was in the process of being determined, and could be purchased in large quantities. As it turned out, kinetic studies of each enzyme pro- vided important information, not only about its own specific mech- anism of action, but also about enzyme catalysis in general.

In 1965, I was recruited to the Chemistry Department at Cornell University by Harold Scheraga, who became a lifelong friend, an invaluable colleague, and a scientific collaborator. Cornell Univer- sity proved to be an ideal place for carrying out interdisciplinary research in biology and chemistry, and its stimulating intellectual environment was a wonderful place to develop my research pro- gram. This Recollection will be confined to the studies of the two enzymes mentioned above and studies of elementary steps in- volved in protein conformational changes and their implications for understanding enzyme catalysis. The general philosophy for using fast reaction techniques was to be able to study the entire time course of the reaction and thus elucidate all of the signifi- cantly populated intermediates along the catalytic pathway. This dissection of the reaction mechanism into elementary steps pro- vided information about the chemical mechanism and the role of the enzyme in catalysis. Furthermore, the study of model reactions, such as hydrogen bonding and protolytic reactions, provided in- formation about the potential role of these elementary reactions in catalysis. These investigations also significantly advanced theoret- ical considerations for treating complex reaction mechanisms and cooperative processes. The 1960s and 1970s, in fact, proved to be very fruitful years for the study of enzyme mechanisms on a va- riety of different fronts, including steady-state kinetics, fast reac- tion kinetics, and model reactions.

The study of ribonuclease was performed with a variety of model substrates, including pyrimidine 3’-phosphates, pyrimidine 2’: 3’- cyclic phosphates, and 3’,5’-dinucleotides (cf. Hammes, 1968). This involved several excellent postdoctoral associates and grad- uate students, and primarily utilized home-built temperature jump

A golden era for understanding enzyme mechanisms 80 1

and stopped-flow temperature jump methods. Because convenient spectral changes did not occur during catalysis, small changes in pH, coupled to an indicator, were used to monitor the reaction progress. The quality of the data in early experiments was appall- ing in retrospect, but we were delighted to get any results with these new methods. Data analysis consisted of drawing the best fit though a polaroid photograph of the kinetic trace and constructing a first-order plot of the data. Later improvements in optics and computer processing of data did not significantly alter the results. The mechanism that emerged in all cases was the binding of sub- strates at rates close to diffusion controlled, followed by a confor- mational change of the enzyme-substrate complex occumng in the millisecond/lOO-microsecond time region. The actual catalytic event involved proton transfers between the intermediate and the en- zyme, and the very important stereochemistry was elucidated by David Usher, also a faculty member at Cornell. The structure of ribonuclease became available during the course of this work and proved very useful for understanding the nature of the conforma- tional change that is probably due to a “hinge” region of the enzyme, folding the enzyme around the substrate, squeezing out water, and orienting imidazoles appropriately for catalysis. (The structure of ribonuclease was determined in 1967 by Harker and coworkers at Roswell Park and Wyckoff, Richards, and coworkers at Yale.) This brief description does not do justice to the consid- erable man/women years devoted to the research, but will suffice for the purpose of this Recollection.

The reaction of ribonuclease is relatively simple, namely hydrolysis/synthesis of P-0 bonds. The reaction of aspartate amino- transferase is considerably more complex because it involves the interchange of an amino group between amino acids (aspartate and glutamate) and keto acids (oxalacetate and ketoglutarate), and uti- lizes a coenzyme, pyridoxal phosphate, in the process. The reac- tion mechanism involves the transfer of the amino group to pyridoxal, thereby forming pyridoxamine, which transfers the amino group to the appropriate keto acid. Thus, the reaction can be con- veniently studied as the two half reactions involving the aspartate- oxalacetate and glutamate-ketoglutarate pairs. The first studies with the temperature jump method clearly showed the formation of the enzyme-substrate complexes and the interconversion of the aldimine-ketimine intermediates, and permitted the associated rates to be determined (Fasella & Hammes, 1967). From the spectral properties of the intermediates, specific structures could be as- signed. The ultimate resolution of the reaction mechanism was found with the substrate erythro-P-hydroxyaspartate. John Haslam, a postdoctoral associate in my laboratory, found that eight relax- ation times could be associated with this half reaction (Hammes & Haslam, 1969). The rate constants for eight steps could be evalu- ated and the nature of the intermediates defined. Without going into detail, the mechanism involved formation of an initial com- plex between enzyme and substrate, a conformational change, for- mation of a Schiff base with pyridoxal, interconversion of the Schiff base to a quinone intermediate, formation of the ketimine, and hydrolysis of the ketimine via a conformational change and an enzyme-substrate complex. I have sometimes referred to this as the world’s record for kinetic intermediates in an enzyme mecha- nism, but admit that I have not researched this claim.

Of course, many other studies of enzyme mechanisms occurred during this period, but this is not intended as a review of the body of excellent work that was published. I would, however, like to mention the work of George Hess, a colleague at Cornell, whose laboratory very beautifully elucidated the elementary steps in the

hydrolysis of amides by chymotrypsin (Hess, 1970). I would be remiss in not mentioning that many organic and physical chemists turned to enzyme mechanisms during this period, and many bio- chemists became much more molecular and mechanism oriented. This interest was enhanced by the determination of an increasing number of enzyme structures by X-ray crystallography. Also, de- termination of the stereochemical course of reactions assisted greatly in postulating detailed mechanisms.

Consideration of the body of data produced from the study of enzyme kinetics with fast reaction methods produced some impor- tant generalizations about how enzymes work. First, in many cases, the initial formation of the enzyme-substrate complex occurs at a rate close to the diffusion controlled limit, with a specific rate constant of about 10’ M” s-’ . (The calculation of this limit was a major part of my Ph.D. thesis.) The lower bound for this rate constant obtained from steady-state kinetic parameters also is often close to the theoretical limit. Qpically, this rate constant decreases markedly for “poor” substrates. Second, the formation of the enzyme-substrate complex is often followed by a conformational change, presumably to create a hydrophobic environment and ori- ent the catalytic groups precisely. A hydrophobic environment has a number of potential advantages for catalysis: for example, the effective dielectric constant is reduced, thereby strengthening elec- trostatic interactions, the pKs of ionizable groups can be signifi- cantly altered, and proton transfer can occur very efficiently.

One of the triumphs of relaxation methods was the ability to study the kinetics of reactions that were previously considered immeasurably fast. From the standpoint of enzymes, the most im- portant of these reactions were proton transfer, hydrogen bonding, and the disruption of water structure. These studies permitted up- per bounds to be put on the rates of acid-base catalysis in enzymes and protein conformational changes. The rate of proton transfer effectively limits the turnover number to 103-105 s-’ for enzymes that utilize acid-base catalysis. The limiting rate for protein con- formational changes is approximately lo9 s - ’ . The fact that most conformational changes are much slower than this is because they are usually highly cooperative, involving multiple (probably hun- dreds of) noncovalent interactions. A great effort was expended by many groups, Rufus Lumry at Minnesota and my own, for exam- ple, to measure the rate of a helix-coil transition in polypeptides. We ultimately placed this rate at about 107-10s s-I. In the last few years, improved experimental techniques have permitted more direct studies of these important transitions.

The occurrence of conformational changes in proteins was pos- tulated and known for many years, at least as early as the 1940s. The role of these conformational changes in enzyme catalysis has been the source of considerable and continuing speculation. It is clear that the macromolecular nature of enzymes is essential- attempts to synthesize small molecules with comparable catalytic efficiency have been singularly unsuccessful. The results from fast reaction studies suggest that enzyme mechanisms generally pro- ceed via multiple intermediates that are significantly populated. In other words, part of the catalytic efficiency is achieved by dividing the catalysis into multiple steps, each step having a considerably lower activation energy than that of the overall uncatalyzed step. These intermediates can involve noncovalent interactions with the enzyme or can involve covalent transfers to coenzymes or en- zymes. A number of years later, the importance of multiple inter- mediates was reformulated in terms of evolutionary pressures, but such considerations are not necessary to appreciate the concept of multiple intermediates enhancing the catalytic efficiency. The im-

802 G.G. Hammes

portance of a macromolecular framework and many intramolecular interactions is consistent with multiple intermediates because the macromolecule can readjust and optimize its structure for each step in the mechanism.

Many people were wrestling with the question of how enzymes achieve their catalytic efficiency. Tom Bruice and coworkers demonstrated convincingly in model systems that restriction of substrate rotational conformers was important, and Bill Jencks developed a theoretical framework based on this idea in terms of entropy concepts. The initial conformational change accompany- ing substrate binding was also described as an induced fit by Dan Koshland, whereby the substrate first binds to an open form of the enzyme, followed by a shift in which the “fit” of the two partners is enhanced. All of these models can be understood in terms of similar concepts: the binding of substrate to the en- zyme induces structural changes that enhance the interaction of enzyme and substrate, restrict the orientation of the substrate to a conformation favorable for catalysis, and alter the environment relative to bulk water. Although there is common agreement about the importance of conformational changes, it is important to mention that the conformational changes observed by X-ray crystallography accompanying substrate binding are generally very small.

The enzyme also may play a more dynamic role in catalysis. For example, Henry Eyting and Rufus Lumry postulated a “rack” model, whereby the enzyme held the substrate and pulled it apart as in the medieval torture chamber. The idea that the protein is able to distort the substrate structure, thereby bringing it closer to the transition state, is still often discussed. A more dynamic role for the protein is that alterations in the substrate are coordinated with simultaneous breaking and formation of multiple noncovalent in- teractions in the protein, effectively causing a lowering of the activation energy. We tried to find experimental evidence for such effects through the use of ultrasonics without success. This idea has resurfaced with the use of a much sharper experimental tool, nuclear magnetic resonance.

The elucidation of elementary steps in enzyme reactions and relevant model systems considerably enhanced the understanding of how enzymes work. Yet, the popularity of such studies waned in the 1970s. I found that the questions surrounding enzyme regula- tion, enzyme-coupled ion transport, and multienzyme complexes were more intriguing. Part of this interlude was due to the diffi- culty of obtaining many enzymes in large quantities and the need for new experimental tools. Happily, both of these situations have been remedied in the 1990s. With the advent of cloning, virtually any enzyme can be obtained in sufficient quantities for transient kinetic studies. Furthermore, the large numbers of molecular struc- tures that are known and the ability to perform site-specific mu- tagenesis permit new types of experiments to be done that could not be contemplated in the 1960s and 1970s. The use of NMR, lasers, and other new methods permit the dynamics of protein conformational changes to be studied from an entirely fresh view- point. Finally, the sophisticated advances in theory may provide a much deeper understanding of the underlying molecular inter- actions and processes. I have often described the time period of my recollections as a golden era for the elucidation of enzyme mech- anisms in terms of their elementary steps. Yet, I am excited by a new era of enzymology, which promises great advances in under- standing the molecular details of enzyme mechanisms.

References

Fasella P, Hammes GG. 1967. A temperature jump study of aspartate amino- transferase: A reinvestigation. Biochemistry 6 : 1798-1804.

Hammes GG. 1968. Relaxation spectrometry of enzymatic reactions. Acc Chem Res 1 3 - 3 2 9 .

Hammes GG, Haslam JL. 1969. A kinetic investigation of the interaction of erythro-;gb-hydroxyaspartic acid with aspartate aminotransferase. Biochem- istry 8:1591-1598.

Hess GP. 1970. Chymotrypsin-Chemical properties and catalysis. In: Boyer P, ed. The enzymes 3rd ed. pp 213-248.

Peller L, Alberty RA. 1959. Multiple intermediates in steady state kinetics. 1. The mechanism involving a single substrate and product. J Am Chem SOC 815907-5914.