ScienceWeek

BIOCHEMISTRY: ENZYME CATALYSIS

The following points are made by S.J. Benkovic and S. Hammes-Schiffer (Science 2003 301:1196):

1) The fascinating catalytic process executed by enzymes has long remained a mystery. How do enzymes achieve accelerated rates for difficult chemical transformations and exquisite specificity toward substrates distinguished only by their stereochemistry?

2) Much has been written about the historical exploration of enzymatic catalysis. Among the first hypotheses offered are the familiar "lock and key" model (Fischer, 1894), which proposed that the binding of a substrate molecule to the active site on the enzyme results in activation of the substrate (in modern terms, a reactive conformation), and a later modified version in which the "key does not quite fit the lock perfectly but exercises a certain strain on it" (Haldane, 1930) (in modern terms, ground-state destabilization). With the advent of transition-state theory, the hypothesis of enzyme-transition-state complementarity (Pauling, 1948), which found a preferential binding of the transition state rather than the substrate or product as the source of catalysis, took center stage. This prediction was neatly satisfied by the first enzyme structure solved, that of lysozyme, with the polysaccharide (N-acetylglucosamine)3 bound at its active site. The structure showed the transition state for glycoside cleavage to be stabilized by the enzyme: The strong electrostatic field of the two carboxylates contributed by Asp52 and Glu35 on either side of the active-site cleft are positioned to interact with the developing positive charge on the oxocarbenium ion.

3) The comparison of enzyme-catalyzed and noncatalytic rates has provided an estimate of the degree of enzymatic transition-state stabilization. Careful measurements of the rates for spontaneous hydrolysis of ionized phosphate monoesters and diesters relative to Escherichia coli alkaline phosphatase or staphylococcal nuclease acting on the same substrate reveals that these enzymes enhance the rates of the hydrolysis reaction by 10^(15)-fold to 10^(17)-fold. These values are at the upper end of rate enhancements and provide a measure of what is meant by a catalytic process.

4) In this picture of catalysis, which takes advantage of thermodynamic state function descriptors of the free energy of activation for the substrate and transition states, enzymic catalytic power will always appear as a result of increased transition-state stabilization (lower free energy) for the enzymic process relative to the reference reaction. How it is parceled among specific forces between the substrate and enzyme, i.e., electrostatic, steric, hydrogen-bonding, or differential solvation effects, is not specified. As a consequence, there has been an increased scrutiny of how the binding interactions arising from favorable and unfavorable noncovalent bonding between the reactants and residues within the active site are translated into catalysis.

Science http://www.sciencemag.org

--------------------------------

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

ScienceWeek http://scienceweek.com