ScienceWeek

BIOCHEMISTRY: ON ENZYME CATALYSIS

The following points are made by M. Garcia-Viloca et al (Science 2004 303:186):

1) Enzyme catalysis, which can produce rate accelerations as large as a factor of 10^(19) (1), involves molecular recognition at the highest level of development. The catalysis of many proton-transfer reactions, for example, requires the recognition of a change in a CH bond length of about 0.5 in going from the reactant to the transition state. In 1946, before structural information was available, Linus Pauling (1901-1994) proposed (2) that enzymes can accelerate rates because they bind the transition state better than the substrate and thereby lower the activation energy.

2) This key concept in enzyme catalysis can now be augmented by a detailed description of the sources of enzymatic rate enhancements based on developments in transition state theory, the availability of structural, kinetic, and thermodynamic data, and the insights provided by computer simulations. An increasing number of articles propose a variety of origins for enzyme catalysis described by terms such as correlated conformational fluctuations, dynamical and nonequilibrium effects, electrostatic pre-organization, entropic guidance, fluctuating barrier height, near-attack configurations, reactant destabilization, and tunneling.

3) In an insightful paper published in 1978 (3), which is as valid today as it was then, Schowen wrote, in accord with Pauling's original insight, "...the entire and sole source of catalytic power [of enzymes] is the stabilization of the transition state..." The authors introduce modern concepts of transition state theory that led to a somewhat modified and more detailed description of the role of the transition state and use it as the framework for describing our present understanding of how enzymes "work". The authors' restatement of the key premise is that "the entire and sole source of the catalytic power of enzymes is due to the lowering of the free energy of activation and any increase in the generalized transmission coefficient, as compared to that of the uncatalyzed reaction."

4) In summary: Advances in transition state theory and computer simulations are providing new insights into the sources of enzyme catalysis. Both lowering of the activation free energy and changes in the generalized transmission coefficient (recrossing of the transition state, tunneling, and nonequilibrium contributions) can play a role. A framework for understanding these effects is presented by the authors, and the contributions of the different factors, as illustrated by specific enzymes, are identified and quantified by computer simulations.(4,5)

References (abridged):

1. R. Wolfenden, M. J. Snyder, Acc. Chem. Res. 34, 938 (2001)

2. L. Pauling, Chem. Eng. News 24, 1375 (1946)

3. R. L. Schowen, in Transition States of Biochemical Processes, R. D. Gandour, R. L. Schowen, Eds. (Plenum, New York, 1978), p77.

4. An historical overview of mechanistic enzymology, which serves as an excellent complement to the present analysis, was published recently by Bugg (2001). He points out that many enzymologists continue to search for the "elusive additional mechanisms by which enzymes may achieve high rates of catalysis". The thesis of the present article is that modern simulations of transition states are a powerful tool for discovering these mechanisms and that all such mechanisms can be understood in terms of various contributions to a specific equation from transition state theory relating the rate constant for a reaction as a function of the temperature.

5. H. Eyring, A. E. Stern, Chem. Rev. 24, 2 (1939)

Science http://www.sciencemag.org

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ENZYME DYNAMICS DURING CATALYSIS

The following points are made by E. Eisenmesser et al (Science 2002 295:1520):

1) Although classical enzymology together with structural biology have provided profound insights into the chemical mechanisms of many enzymes (1), enzyme dynamics and their relation to catalytic function remain poorly characterized. Because many enzymatic reactions occur on time scales of micro- to milliseconds, it is anticipated that the conformational dynamics of the enzyme on these time scales might be linked to its catalytic action (2). Classically, enzyme reactions are studied by detecting substrate turnover.

2) Dynamics of enzymes during catalysis have been detected with methods such as fluorescent resonance energy transfer, atomic force microscopy, and stopped-flow fluorescence, which report on global motions of the enzyme or dynamics of particular molecular sites. In contrast, nuclear magnetic resonance (NMR) spectroscopy enables investigations of motions at many atomic sites simultaneously (3,4). NMR studies reporting on the time scales, amplitudes, and energetics of motions in proteins have provided information on the relation between protein mobility and function (5).

3) The authors report an examination of enzyme catalysis in a nonclassical way by characterizing motions in the enzyme during substrate turnover. The authors have used NMR relaxation experiments to advance these efforts by characterizing conformational exchange in an enzyme, human cyclophilin A (CypA), during catalysis. CypA is a member of the highly conserved family of cyclophilins that are found in high concentrations in many tissues. Cyclophilins are peptidyl-prolyl cis/trans isomerases that catalyze the interconversion between cis and trans conformations of X-Pro peptide bonds, where "X" denotes any amino acid. CypA operates in numerous biological processes. It is the receptor for the immunosuppressive drug cyclosporin A, is essential for HIV infectivity, and accelerates protein folding in vitro by catalyzing the rate-limiting cis/trans isomerization of prolyl peptide bonds. However, its function in vivo and its molecular mechanism are still in dispute.

4) In summary: Internal protein dynamics are intimately connected to enzymatic catalysis. However, enzyme motions linked to substrate turnover remain largely unknown. The authors have studied dynamics of an enzyme during catalysis at atomic resolution using nuclear magnetic resonance relaxation methods. During catalytic action of the enzyme cyclophilin A, the authors detect conformational fluctuations of the active site that occur on a time scale of hundreds of microseconds. The rates of conformational dynamics of the enzyme strongly correlate with the microscopic rates of substrate turnover. The authors suggest that the present results, together with available structural data, allow a prediction of the reaction trajectory.

References (abridged):

1. T.C. Bruice and S.J. Benkovic, Biochemistry 39, 6267 (2000)

2. A. Fersht, Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding (Freeman, New York, ed. 1, 1999) pp. 44-51

3. M.W. Fischer, A. Majumdar, E. R. P. Zuiderweg, Progr. Nucl. Magn. Reson. Spectrosc. 33, 207 (1998)

4. A.G. Palmer, 3rd, Curr. Opin. Struct. Biol. 7, 732 (1997)

5. R. Ishima and D.A. Torchia, Nature Struct. Biol. 7, 740 (2000)

Science http://www.sciencemag.org

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CRYSTAL STRUCTURE OF A HAIRPIN RIBOZYME INHIBITOR COMPLEX WITH IMPLICATIONS FOR CATALYSIS

The following points are made by P.B. Rupert et al (Nature 2001 410:780):

1) The hairpin ribozyme is a catalytic RNA derived from the self-cleaving and ligating domain of the negative polarity strand of the satellite RNA of tobacco ringspot virus. In vivo, this domain is responsible for generating unit-length circular satellite RNA during the course of its rolling-circle replication (1). The cleavage reaction generates products with 5'-hydroxyl and 2',3'-cyclic phosphate termini, which are analogous to those produced by three other natural ribozymes that are part of circular satellite RNAs: the hammerhead, the hepatitis delta virus (HDV) and the Varkud satellite (VS) ribozymes. These four ribozymes, however, are structurally unrelated, and therefore represent independent evolutionary solutions to the same biochemical problem (2). None of these ribozymes require proteins for activity. They are also quite small, for example the hairpin ribozyme requires about 50 nucleotides for activity. The hairpin ribozyme is an ideal experimental system to help understand how a small catalytic RNA self-assembles and forms an active site.

2) Biochemical experiments demonstrated that the active site of the hairpin ribozyme results from the association of two largely helical segments of the satellite RNA, stems A (one strand of which contains the scissile phosphate) and B. Both contain nucleotides necessary for catalysis(1). The stems can be synthesized as two separate RNAs and mixed in vitro to reconstitute an active ribozyme(3,4). Most biochemical experiments have been carried out using constructs where the two stems are connected by a single-stranded linker. In the satellite RNA, stems A and B are part of a four-helix junction(5). Fluorescence resonance energy transfer (FRET) measurements demonstrated that the docking of stems A and B is greatly favoured in constructs that contain the four-helix junction, compared with systems where the two stems are connected by an extended linker(5).

3) In summary: The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbors the scissile bond. The 2.4 angstrom resolution structure of a hairpin ribozyme inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.

References (abridged):

1. Fedor, M. J. Structure and function of the hairpin ribozyme. J. Mol. Biol. 297, 269-291 (2000)

2. McKay, D. B. & Wedekind, J. E. in The RNA World (eds Gesteland, R. F., Cech, T. R. & Atkins, J. F.) 265-286 (Cold Spring Harbor Press, Cold Spring Harbor, 1999).

3. Butcher, S. E., Heckman, J. E. & Burke, J. M. Reconstitution of hairpin ribozyme activity following separation of functional domains. J. Biol. Chem. 270, 29648-29651 (1995)

4. Shin, C. et al. The loop B domain is physically separable from the loop A domain in the hairpin ribozyme. Nucleic Acids Res. 24, 2685-2689 (1996)

5. Murchie, A. I. H., Thomson, J. B., Walter, F. & Lilley, D. M. J. Folding of the hairpin ribozyme in its natural conformation achieves close physical proximity of the loops. Mol. Cell 1, 873-881 (1998)

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