Examining the behavior of substrates for staphylococcal nuclease and orotidylate decarboxylase, we have determined the half-times ofthe corresponding reactions in the absence of a catalyst, under ambient conditions, are 130,000 years and 78,000,000 years respectively (A Proficient Enzyme, A. Radzicka and R. Wolfenden, Science 267, 90, 1995). For the corresponding enzyme reactions, these values imply that the Ki values of ideal transition state analogue inhibitors are 10-20 M and 10-23 M, respectively. The sources of these very high affinities are a source of rational curiosity.
To learn more, we are analyzing the interactions of the corresponding enzymes with transition state analogue inhibitors by X-ray crystallography and heavy atom NMR, combined with analyzing the kinetic results of site-directed mutagenesis.
Highlights of current research include P. Shih's elucidation of the forms of substrates and products bound by adenosine and cytidinedeaminases (Biochemistry 35, 3697, 1996); demonstration by S. Bearne and D. Carlow of high levels of cooperativity in the action ofbinding determinants at the active sites of several enzymes, using site-directed mutagenesis and chemical modification (Biochemistry36, 1646-1656, 1997); establishment of the nonenzymatic rates of cleavage of internal, C-terminal and N-terminal peptide bonds inproteins (JACS 118, 6105, 1996), the phosphodiester bonds of DNA (JACS 120, 833-834, 1998), and the glycosidic bonds of starch andcellulose (JACS 120, 6814, 1998).
As one of the factors that must be considered in efforts to analyze "molecular recognition", wehave also been determining the strengths and directional preferences of interactions of biological molecules with solvent water, mostrecently those of the nucleic acid bases.
(J. Mol. Biol. 280, 421, 1998)
There has been widespread interest in the mechanism of action of OMP decarboxylase, which reduces the half-time for decarboxylation of orotidine 5'-phosphate from 78 million years to 40 milliseconds. In the spring of 2000, the crystal structure of this enzyme was reported by 4 research groups almost simultaneously, including our own (Proc. Natl. Acad. Sci. 97, 2011, 2000). These structures, involving proteins from 4 different organisms in complex with substrate and transition state analogues, revealed a major surprise.
Not a single one of the complex mechanisms that had been proposed previously by various investigators (including prominent theoreticians) appeared to be correct. Instead, this enzyme appears to operate by a disarmingly simple mechanism that involves stabilization of a carbanion by an amino group at the enzyme's active site. By examining the effects on activity of mutating various groups at the active site, we are beginning to gain an appreciation of the extraordinary levels of cooperativity that are involved in the action of this enzyme.
(Biochemistry 39, 8113, 2000)