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- Publisher
- American Society for Biochemistry and Molecular Biology
- Copyright
- Copyright © 2006 Elsevier Inc.
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- DOI
- 10.1074/jbc.x600001200
- Publisher site
- See Article on Publisher Site
Abstract
This paper is available online at www.jbc.org REFLECTIONS THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 10, pp. 6117–6119, March 10, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. A PAPER IN A SERIES COMMISSIONED TO CELEBRATE THE CENTENARY OF THE JBC in 2005 JBC Centennial 1905–2005 100 Years of Biochemistry and Molecular Biology Mechanistic Inferences from Stereochemistry PUBLISHED, JBC PAPERS IN PRESS, FEBRUARY 1, 2006, DOI 10.1074/JBC.X600001200 I. A. Rose From the Department of Physiology and Biophysics, University of California School of Medicine, Irvine, California 92717 am still impressed that the textbook language of organic chemistry is so successful in explaining the reactions that we find in nature. This might not have been anticipated because we find that biological reactions are invariably stereospecific whereas nonbiological reac- tions are invariably not. This realization came to light about 150 years ago when Pasteur found that only half of chemically prepared tartarate was fermentable contrary to chemically identical tartarate from grapes. Only the natural product rotated the plane of polarized light (1). A new geometry had to be found for carbon to explain this. It could not be planar. As understood by Ogston in 1948 (2) the stereospecificity of biological reactions derives from the chiral properties of the active sites of the enzymes that catalyze them. (Pasteur realized that only a chiral reagent would be able to distinguish between substrates that were not superimposable on their mirror images. He spent much effort searching for a natural force that might have caused chirality in the first place. It remains an unsolved problem.) When I began studying simple enzymatic reactions at carbon centers in 1955, I was not at all sure what I might run into. Organic chemists were acquiring evidence for stable ion pair interme- diates in carbonium and carbanion rearrangements in solution that were completely stereospe- cific. Would the stereospecificity of enzymatic reactions turn out to be explained in terms of a physical or chemical role of the enzyme? If this seems like an overstatement it may be recalled that in 1955 there were no examples to cite in which an enzyme could be analyzed to be acting as a base to abstract a proton from –CH to a carbonyl. However, there were well known enzymes available such as the aldolases and aldose-ketose isomerases, the mechanisms of which had not been ana- lyzed. My hope then was to use a stereochemical approach to the study of enzyme reaction chemistry as my first research problem. Fortunately scintillation counters were becoming available in 1955. I was somewhat ahead of the game because of the hobby of Seymour Lipsky, an M.D. in the Department of Medicine who consulted for the New Haven-based Technical Measurement Co. and had one of their first com- mercial counters. Tritium, especially T-water, was also becoming available, making it unnecessary for me to repair the Rittenberg model mass spectrometer that Henry Hoberman had left to the Yale Department of Biochemistry. The first demonstration of an enzyme acting as a base was probably our observation in 1955 that muscle aldolase catalyzed the stereospecific exchange of one of the C-1 hydroxymethyl protons of dihydroxyacetone-P with TOH in the absence of an aldehyde partner (3). From the stereochem- istry of the T-exchanged product (4) compared with that of C-4 of the condensation products one MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6117 This is an Open Access article under the CC BY license. REFLECTIONS: A Portrait of RIO Kinases could conclude that the aldehyde and the proton must general picture and extended it in great detail (11). The approach the stable intermediate from the same direction. simple single acid/single base mechanism may have pro- Evidence that “proton abstraction” is the result of transfer vided the evolutionary pressure that seems to favor this to a stable position on the enzyme and not to an anionic mechanism. group on substrates comes from a number of examples in Because pentoses and hexoses must be acted upon in which intermolecular proton transfer could be shown. For their far more abundant cyclic forms it was of interest to example, with fumarase the reacting tritium derived from ask if the anomeric specificity of each isomerase could be malate can be rescued from exchange with solvent by car- predicted from the topology of its E-H cis-enediol inter- rying out the reaction in the presence of fluorofumarate. In mediate (9, 12). When C-2 becomes tetrahedral by proton fact the loss of the proton from free enzyme is the slowest transfer from one face of the enediol, the ring-closing OH step of the reaction cycle. of C-4 (for a pentose) or of C-5 (for a hexose) would only In 1957 the conversion of fructose-6-P to glucose-6-P in be able to reach C-1 from the opposite face. The predic- D O had been reported to occur with little or no transfer tions made on this basis proved to be correct in all five of the substrate hydrogen to the neighboring carbon of the cases that have been examined (8). product by phosphoglucose isomerase (PGI) (5). The Absolute stereochemistry often obscures the details of absence of transfer might indicate that the enzyme did not an enzyme’s reaction mechanism. In the classic experi- act as a base or that different bases were used for C-1 and ments of Bender (13, 14) the tetrahedral gem-diol inter- C-2. We inadvertently discovered that the PGI result was mediate formed in the base-catalyzed hydrolysis of esters misleading, caused by inadequate trapping of the product was readily detected by back exchange of O between that led to its redundant exchange with the medium. In the water and the recovered ester. However, an enzymatic Glc-6-P to Fru-6-P direction in D O we found a surprising reaction with a comparable mechanism would not be overshoot of the equilibrium that was seen when the Fru- expected to show exchange because the reversal of the 6-P was determined by a color test but not when deter- hydration step would remove the same oxygen that was mined by an enzymatic assay that went to completion (6). introduced in forming the intermediate. Only if the inter- The colorimetric assay typically does not go to completion mediate was able to interchange the identical groups and therefore is sensitive to a kinetic deuterium isotope (positional exchange, not possible for C in this case) could effect. The first Fru-6-P to be formed had derived some of one expect otherwise. its proton from the substrate as hydrogen. At later times Middlefort and Rose (15) used positional isotope this Fru-6-P acquired deuterium by back exchange with exchange to establish the formation of glutamyl-P as an the solvent giving a lower color equivalent with time intermediate in the glutamine synthetase reaction: gluta- although the reaction was already close to equilibrium. mate ATP NH 3 glutamine ADP P.A 3 i Hence the apparent overshoot (6). Both T-transfer and two-step mechanism, E glutamate ATP 7 E- T-exchange were found to occur in a single turnover when ADP-glutamyl-P 7 E glutamine P , was contraindi- the reaction was run in the opposite direction with a good cated by failure to observe ATP:ADP exchange unless NH trap for the product: 1T-Fru-6-P3 Glc-6-P3 6-P-gluco- was also present. However, this could have been due to nate. Thus was established the formation of an exchange- tightly bound ADP. We realized that even tightly bound sensitive E-T enediol intermediate. Transfer was shown to ADP might be able to achieve torsional equilibration of its be intramolecular (7). The stereochemistry of C-1 of phosphoryl oxygens, in which case the - bridge O of 1T-Fru-6-P and the intramolecular nature of the transfer ATP would scramble into non-bridge positions of reiso- establish that the enediol was of the cis configuration (8). lated ATP if ATP:ADP exchange proved to be the case. The same stereochemistry was found for all seven isomer- Indeed, positional isotope exchange was found in the ases that we examined (9). Only xylose isomerase showed absence of NH at a rate greater than required from the no exchange and is believed to be a hydride transfer maximum rates of the forward and reverse net reactions reaction. (16). The generally observed cis-enediol mechanism allows a Such is my confidence that all enzymatic reactions are unique acid group to polarize either the C-1 or the C-2 stereospecific that finding one otherwise suggests that a carbonyl of the substrates. Because nucleophilic attack by nonenzymatic step must be part of the reaction sequence. epoxides is acid-catalyzed we anticipated their value in the As an example, the enzyme called methylglyoxal (MG) active site labeling of isomerases (10). Subsequent struc- synthase actually produces enol-pyruvaldehyde (ePy), not tural studies of triose-P isomerase have confirmed our MG, from dihydroxyacetone-P. The MG arises from 6118 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 REFLECTIONS: A Portrait of RIO Kinases tero-PEP that allowed us to study the chirality of reactions with PEP and ketonization of the product ePy in solution. This was enolpyruvate; Jacob bar Tana for help in developing the pulse-chase anticipated when the –CH group of the MG was found to method for determining the functionality of enzyme-substrate com- have been made nonstereospecifically (17). The ketoniza- plexes; C. F. Middlefort for creative work in analyzing the distribution of O in ATP in the PIX study of glutamine synthetase; John Richard who tion of ePy is a slow step for which no enzyme has been alerted me to the inherent instability of 1,2-propenediol-3-P, which I had found. Instead, glutathione adds spontaneously to the car- tried to generate for mechanism studies; and Avram Hershko for many bonyl of the ePy, and the rapidly ketonized adduct years of fruitful collaboration in the area of ubiquitin-dependent protein becomes the substrate for reaction with glyoxalase I (18). breakdown. Thanks are also due to Edward O’Connell and Jessie Warms for excellent technical assistance. Why the synthase did not evolve to carry out the keton- ization step is a puzzle because there is a very active Address correspondence to: [email protected] enzyme, glyoxalase III, that would utilize MG directly. Per- haps it has something to do with the fact that methyl- REFERENCES glyoxal is fairly toxic, forming stable complexes with 1. Mason, S. F. (1991) Chemical Evolution, pp. 260–284, Clarendon Press, Oxford 2. Ogston, A. G. (1948) Nature 162, 963 proteins. 3. Rose, I. A., and Rieder, S. V. (1955) J. Am. Chem. Soc. 77, 5764–5765 For an excellent presentation see Stereochemistry and 4. Rose, I. A. (1958) J. Am. Chem. Soc. 80, 5835–5836 Its Application to Biochemistry by William L. Alworth, 5. Topper, Y. J. (1957) J. Biol. Chem. 225, 419–426 6. Rose, I. A. (1962) Brookhaven Symp. Biol. 15, 293–309 John Wiley & Sons, Inc., New York (1972). Autobiograph- 7. Rose, I. A., and O’Connell, E. L. (1961) J. Biol. Chem. 236, 3086–3092 ical presentations relating to subjects discussed in this 8. Rose, I. A., and O’Connell, E. L. (1960) Biochim. Biophys. Acta 42, 159–160 9. Rose, I. A. (1975) Adv. Enzymol. 43, 491–517 paper can be found in Protein Science (1995), pp. 1430– 10. O’Connell, E. L., and Rose, I. A. (1977) Methods Enzymol. 46, 381–388 1433 and in “Les Prix Nobel, 2004,” pp. 203–217. 11. Davenport, R. C., Bash, P. A., Seaton, P. A., Karplus, M., Petsko, G. A., and Ringe, D. (1991) Biochemistry 30, 5821–5826 12. Schray, K. J., Benkovic, S. J., Benkovic, P. A., and Rose, I. A. (1973) J. Biol. Chem. Acknowledgments—I have been very fortunate in collaborations with 248, 2219–2224 many skilled colleagues. Some of the more significant of these have been: 13. Bender, M. L. (1951) J. Am. Chem. Soc. 73, 1626–1629 Kenneth Hanson, whose scholarship provided the idea for doing the 14. Bender, M. L., and Heck, H. (1967) J. Am. Chem. Soc. 89, 1211–1220 chiral specificity of citric acid; Lindo A. Patterson, for the idea and guid- 15. Middlefort, C. F., and Rose, I. A. (1976) J. Biol. Chem. 251, 5881–5887 ance in doing the absolute structure of chiral 2-deutero-glycolate by neu- 16. Rose, I. A. (1978) Fed. Proc. 37, 2775–2782 tron diffraction crystallography, allowing us to confirm chirality of many 17. Summers, M. C., and Rose, I. A. (1977) J. Am. Chem. Soc. 99, 4475–4478 reactions; Mildred Cohn, for the NMR determination of specific 3-deu- 18. Rose, I. A., and Nowick, J. S. (2002) J. Am. Chem. Soc. 124, 13047–13052 MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6119
Journal
Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology
Published: Mar 10, 2006