178 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Ace. zyxwvuts Chem. Res. zyxwv 1995,28, zyxwv 178-186 Mandelate Racemase: Structure-Function Studies of a Pseudosymmetric Enzyme GEORGE L. KENYON,*>’ JOHN A. GERLT,$ GREGORY A. PETSKO,~ AND JOHN W. KOZARICH~~ Department of Pharmaceutical Chemistry, University of California, San Francisco, California zyxw 94143, Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, The Rosenstiel Center, Brandeis University, Waltham, Massachusetts 02254, and Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065 Received October 14, 1994 Introduction Racemases (and their close cousins, the epimerases) have always intrigued biochemists because they must process both substrate enantiomers (or diastereoiso- mers in the case of epimerases). zyxwvuts So the question arises, how can enzymes, which are inherently asym- metric, deal with both enantiomers with at least approximately equal facility? Most enzymes, after all, are famous for being exquisitely stereoselective. But racemases by definition have equilibrium constants equal to unity and therefore, according to the Haldane relationship, h,,JK, in one direction must be equal to k,,JK, in the reverse direction. The logical answer to this question is that racemases must have evolved a “functional asymmetry” or “pseudosymmetry” in George L. Kenyon was born in Wilmington, DE, in 1939. He received his B.S. in chemistry from Bucknell and his A.M. and Ph.D. degrees in organic chemistry from Halvard, working with Professor F. H. Westheimer. After an NIH postdoctoral fellowship in biochemistry at MIT with Professor J. M. Buchanan (1965-1966), he joined the faculty at the University of California, Berkeley, as assistant professor of chemistry. He moved to UCSF in 1972, where he is currently professor of chemistry and pharmaceutical chemistry and dean of the School of Pharmacy. He has wide- ranging research interests that include studies of the mechanisms of enzymatic reactions. applications of recombinant DNA technology to enzymology, and the development of structure-based approaches to the rational design of both enzymatic inhibitors and nucleic acid binding agents as potential drugs. With his wife, Lucy, he also enjoys the observations of avifauna on a global scale, and in this regard, he has been known at times to be considered a ‘Ywitcher“ John A. Gerlt was born in Sycamore, IL, in 1947. He earned his B.S. degree in biochemistry from Michigan State University (1969) and his A.M. (1970) and Ph.D. (1974) degrees in biochemistry and molecular biology from Haward University, where he studied with Professor Frank H. Westheimer. His postdoctoral studies, supported by a Jane Coffin Childs Memorial Fund Fellowship, were with Dr. Christian B. Anfinsen at the National Institutes of Health, Bethesda, MD (1974-1975). He has held positions as assistant and associate professor of chemistry at Yale University (1975-1984) and as professor of chemistry and biochemistry at the University of Maryland, College Park (1984-1994). He recently moved to the University of Illinois, Urbana- Champaign, where he is professor and head of biochemistry. His research interests are focused on understanding the relationships among rates, mechanisms, and active site architectures of various enzyme-catalyzed reactions. His spare time is spent with his wife Jennifer enjoying salt water fish, both in his tank at home and on the coral reefs of St. John. In addition to the fish, his pets include a dog named Eminence and cats named Frank and George. Gregory A. Petsko was born in Washington, DC, in 1948. He received his A.B. from Princeton University and his D.Phil. from Oxford University, where he did his thesis research with Sir David Phillips. After a brief stint with Professor Pierre Douzou in Paris, he joined the faculty of Wayne State University School of Medicine as instructor in biochemistry, becoming assistant professor in 1976. He moved to MIT in 1979 as associate professor and later (1985) professor of chemistry. In 1990 he moved to Brandeis University, where he is now Lucille P. Markey Professor of Biochemistry and Chemistry and director of the Rosenstiel Basic Medical Sciences Research Center. His research interests include the structural basis of the catalytic power of enzymes, the functional roles of protein dynamics, hyperthermostability, and the structural enzymology of mRNA turnover. His nonscientific diversions include old books, old movjes, old cars, and old Scotch. John W. Kozarich is executive director of biochemistry at Merck Research Laboratories in Rahway, NJ. After graduation from Boston College (1971), he completed his Ph.D. research in biological chemistry at MIT with Sidney Hecht (1975) and did a postdoctoral in biochemistry at Halvard with Jack Strominger. He has been professor of pharmacology at Yale University School of Medicine and professor of chemistry and biochemistry at the University of Maryland, College Park. An American Cancer Society Faculty Research Award (1983-1988) and the Pfizer Award in Enzyme Chemistry (1988) are his most notable recognitions. His research interests are mechanisms of enzyme and drug action and their application to drug discovery and enzyme catalysis. their active ~ites.l-~ In this Account, we provide considerable insight into this question as we now have an X-ray crystal structure of mandelate racemase and several of its mutational variants at reasonably high res~lution.~-l~ In fact, mandelate racemase is so far the only racemase for which an X-ray crystal structure is available. Another puzzling mechanistic question in enzymol- ogy is, how can enzymes catalyze rapid proton ex- change (and racemizations, isomerizations, or P-elim- inations) involving carbon-hydrogen bond cleavage of carbon acids with relatively high pKa values? The pKa for the a-hydrogen of mandelic acid, for example, has been estimated to be -22 and that for the mandelate anion to be -29.15 The difficulty in removing this proton has been shown experimentally by Pocker,16 who found that sodium mandelate undergoes ex- change of its a-hydrogen in 0.40 M NaOD in DzO only very slowly even at 100 “C. In contrast, enzymatically, this same exchange reaction occurs with a turnover number of -1000 s-l at 25 “C even at pH 7.17 In this Account, some important insights will be presented University of California. F University of Illinois. 4 Brandeis University. ‘I Merck Sharp & Dohme. (1) Wang, E.; Walsh, C. T. Biochemistry 1978, 17, 1313-1321. (2) Wang, E.; Walsh, C. T. Biochemistry 1981, 20, 7539-7546. (3) Whitman, C. P.; Hegeman, G. D.; Cleland, W. W.; Kenyon, G. L. Biochemistry 1985,24, 3936-3942. (4) Kenyon, G. L.; Whitman, C. P. Mechanisms of Enzymatic Reac- tions: Stereochemistry; Frey, P. A,, Ed.; Elsevier, 1986; pp 191-204. zyxwvutsrqp (5) Neidhart, D. J.; Powers, V. M.; Kenyon, G. L.; Ransom, S. C.; Tsou, A. Y.; Gerlt, J. A,; Petsko, G. A. J. Biol. Chem. 1988, 263, 9268-9270. (6) Neidhart, D. J.; Kenyon, G. L.; Gerlt, J. A.; Petsko, G. A. Nature 1990, 347, 692-694. (7) Gerlt, J. A,; Kenyon, G. L.; Kozarich, J. W.; Lin, D. T.; Neidhart, D. C.; Petsko, G. A,; Powers, V. M.; Ranson, S. C.; Tsou, A. Y. Chemical Aspect of Enzyme Biotechnology: Fundamentals: Scott, A. I., Baldwin, T. O., Raushel, F. M., Eds.; Plenum: New York, 1990; pp 9-21. (8) Neidhart, D. J.; Howell, P. L.; Petsko, G. A,; Powers, V. M., Li, R.; Kenyon, G. L.; Gerlt, J. A. Biochemistry 1991, 30, 9264-9273. (9) Landro, J. A,: Kallarakal, A,; Ransom, S. C.; Gerlt, J. A,; Kozarich, J. W.; Neidhart, D. J.; Kenyon, G. L. Biochemistry 1991,30,9274-9281. (10) Gerlt, J. A,; Kozarich, J. W.; Kenyon, G. L.; Neidhart, D. J.; Petsko, G. A. Proceedings of the Ninth International Biotechnology Symposium, Harnessing Biotechnology for the 21st Century: Ladisch, M. R., Bose, A,, Eds.; ACS Symposium Series: Washington, DC, 1992; pp (11) Gerlt, J. A,; Kenyon, G. L.; Kozarich, J. W.; Petsko, G. A.; Powers, V. M. Curr. Opin. Struct. Biol. 1992, 2, 736-742. (12) Landro, J. A,; Gerlt, J. A,; Kozarich, J. W.; Koo, C. W.; Shah, V. J.; Kenyon, G. L.; Neidhart, D. J.; Fujita, S.; Clifton, J. R.; Petsko, G. A. Biochemistry 1994, 33, 635-643. (13) Mitra; B.; Kallarakal, A,; Kozarich, J. W.; Gerlt, J. A,; Clifton, J. R.; Petsko, G. A,; Kenyon, G. L. Biochemistry 1995,34, 2777-2787. (141Kallaraka1, A. T.; Kozarich, J. W.; Gerlt, J. A,; Clifton, J. R.; Kenyon, G. L. Biochemistry 1995,34, 2788-2797. (15) Gerlt, J. A,; Kozarich, J. W.; Kenyon, G. L.; Gassman, P. G. J. Am. Chem. Soc. 1991,113, 9667-9669. (16) Pocker, Y. Chem. Ind. (London) 1958, 1117-1118. (17)Maggio, E. T.; Kenyon, G. L.; Mildvan, A. S.; Hegeman, G. D. 155-159. Biochemistry 1975,14, 1131-1139. 0001-4842/95/0128-0178$09.00/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1995 American Chemical Society