Crystallographic enzyme catalysis

A X-ray crystallographic method for detecting the transient accumulation of intermediates in enzyme catalysis, protein folding, ligand-binding interactions, and other processes involving macromolecules. The approach is premised on the well documented retention of substantial reactivity of biological macromolecules, even in the crystalline state. 

The x-ray crystallographic analysis of protein structures is a remarkably successful technique. Since the publication of the first protein structure, that of myoglobin in 1958, many other protein structures have been determined. The resulting structural details often approaching atomic level have led to great insights into enzyme catalysis, hormone function, the organisation of the immune system, the molecular architecture of virus particles and protein synthesis. Why then should such an apparently successful technique need synchrotron radiation ... 

The following detailed discussion of three enzymes that have metal ions at their active sites will point out the current state of the art of enzymologists understanding of enzymic catalysis. The examples have been chosen to include the most advanced use of stereochemical techniques, kinetic methodology, solution structural data (NMR, EPR, fluorescence energy transfer), and x-ray crystallographic structures. 

Coleman, D. E., and Sprang, S. R. (1999a). Reaction dynamics of G-protein catalyzed hydrolysis of GTP as viewed by X-ray crystallographic snapshots of Gial. In Methods in Enzymology Enzyme Kinetics and Mechanism, Part E Energetics of Enzyme Catalysis (V. L. Schramm and D. L. Puvich, Eds.), Vol. 308, pp. 70-92. Academic Press, New York. 

A similar chelation of metal to enzyme-bound substrate may also contribute to enzyme catalysis of proton transfer at carbon. For example, X-ray crystallographic analysis of complexes bet veen 3-keto-L-gulonate 6-phosphate decarboxylase and analogs of the 1,2-enediolate reaction intermediate provide evidence that the essential magnesium dication is stabilized by coordination to both the C-2 oxygen and the nonreacting C-3 hydroxy of the reaction intermediate [88]. 

A variety of techniques have been applied to investigate enzyme reaction mechanisms. Kinetic and X-ray crystallographic studies have made major contributions to the elucidation of enzyme mechanisms. Valuable information has been gained from chanical, spectroscopic and biochemical studies of the transition-state structures and intermediates of enzyme catalysis. Computational studies provide necessary refinement toward our understanding of enzyme mechanisms. The ability of an enzyme to accelerate the rate of a chemical reaction derives from the complementarity of the enzyme s active site structure to the activated complex. The transition state by definition has a very short lifetime ( 10 s). Stabilization of the transition state alone is necessary but not sufficient to give catalysis, which requires differential binding of substrate and transition state. Thus a detailed enzyme reaction mechanism can be proposed only when kinetic, chemical and structural components have been studied. The online enzyme catalytic mechanism database is accessible at EzCatDB (http //mbs.cbrc.jp/EzCatDB/). 

In order to understand mechanisms of enzyme catalysis not only are the tertiary structures of enzymes of interest hut so too are the tertiary structures of enzyme-suhstrate complexes. There was, however, a problem As incredibly efficient catalysts, enzymes turn over substrate molecules in fractions of a second, while (40 years ago) collection of crystallographic data took days. The answer to the problem was to study complexes of exceedingly sluggish substrates, unreactive model substrates, as well as strongly bound inhibitors that compete for the active site. 

In spite of the mass of existing literature concerned with the mechanisms of enzyme catalysis (more, perhaps, than in the field of physical organic chemistry), in no single case can an answer be given J to the question of how enzymes increase the rates of known chemical reactions by factors which may be as high as 10 or 10 . In one case, the complete three-dimensional structure of a small enzyme (lysozyme (egg-white) mol. wt. 14,600) is now known, as a result of collaborative efforts of protein chemists and crystallographers. Unfortunately, the chemistry of the substrate of this particular enzyme... 

Crystallographic studies (Blow, 1976) of the structure of the enzyme, enzyme-substrate complexes and enzyme-product complexes have identified a common feature in catalysis by the serine protease enzymes such as a-chymotrypsin. This is the well-known charge-relay system (44), in which... 

The expansion in the power of computers and theoretical methods has made it possible to investigate the mechanism of action of enzymes by combinations of quantum-mechanical and molecular-mechanical calculations. A study of two possible mechanisms for dihydrofolate reductase catalysis was consistent with indirect proton transfer from aspartate to N-5 of the pterin as has been suggested for many years by crystallographic evidence <2003PCB14036>. This conclusion is also consistent with the outcome of a study that directly measured the of the active site aspartate in the Lactobacillus casei enzyme <1999B8038>. Observations of chemical shifts of... 

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