Richard Wolfenden, PhD

RESEARCH INTERESTS:

Enzymes allow organisms to channel the flow of matter to their own advantage, allowing some reactions to proceed rapidly compared with other reactions that offer no selective advantage to the organism. After a substrate is bound at an enzyme’s active site, its half-life is usually a small fraction of 1 s. Rapid turnover is necessary if any enzyme is to produce a significant rate of reaction at the limited concentration (<10-5 M) at which enzymes are present within the cell. Many enzymes are known to have evolved to work nearly as efficiently as is physically possible, with second order rate constants that approach their rates of encounter (∼109 M-1s-1) with the substrate in solution. How rapidly would biological reactions occur if an enzyme were not present? Until recently, some reactions were known to require several years, and everyday experience suggests that some reactions are slower still. The survival of paper documents and ancient ships for long periods under water implies that the glycosidic bonds of cellulose, for example, are very resistant to hydrolysis in the absence of cellulases that catalyze their hydrolysis.

Why would one wish to know the rate of a biological reaction in the absence of an enzyme? That information would allow biologists to appreciate what natural selection has accomplished in the evolution of enzymes as proficient catalysts and would enable chemists to compare enzymes with artificial catalysts produced in the laboratory. Such information would also be of value in considering the design of enzyme antagonists: the greater the rate enhancement that an enzyme produces, the greater is its affinity for the altered substrate in the transition state compared with its relatively modest affinity for the substrate in the ground state. That principle has furnished a basis for the design of transition state analogues, extremely powerful inhibitors that resemble the transition state and take advantage of that special affinity. Examples have now been discovered for enzymes of every class, including inhibitors that are already used to control hypertension, the spread of HIV, the maturation of insects and the growth of weeds. By allowing “snapshots” of enzymes in action, transition state analogues have also provided valuable tools for investigating enzyme structures and mechanisms, most recently that of the peptide bond forming center of the ribosome. Those enzymes that produce the largest rate enhancements and transition state affinities should offer the most sensitive targets for inhibitor design.

Particularly remarkable are those enzymes that act as simple protein catalysts, without the assistance of metals or other cofactors. To determine the extent to which one such enzyme, human uroporphyrinogen decarboxylase, enhances the rate of substrate decarboxylation; we examined the rate of spontaneous decarboxylation of pyrrolyl-3-acetate. Extrapolation of first-order rate constants measured at elevated temperatures indicates that this reaction proceeds with a half-life of 2.3 x 109 years, approaching the age of the Earth. This enzyme shows no significant structural or sequence homology with yeast orotidine 5'-monophosphate decarboxylase, another cofactorless enzyme that catalyzes a very slow reaction. To uncover the mechanisms of action of these remarkable molecules, we are studying these and other enzymes by kinetic and structural methods, site-directed mutation and the study of model reactions. In addition to more traditional methods, these projects make extensive use of new methods that include high-field NMR, isothermal calorimetry, and kinetic experiments in water and other solvents in sealed tubes at very high temperatures.

RECENT PUBLICATIONS:

Snider, M. J., Wolfenden, R., Site-Bound Water and the Shortcomings of a Less than Perfect Transition State Analogue, Biochemistry 40, 11364-11371 (2001)

Wolfenden, R., Snider, M. J., The Depth of Chemical Time and the Power of Enzymes as Catalysts, Accounts of Chemical Research 34, 938-945 (2001).

Miller, B. G., Wolfenden, R., Catalytic Proficiency: The Unusual Case of OMP Decarboxylase, Annu. Rev. Biochem. 71, 847-885, 2002.

Lad. C., Williams, N. H., Wolfenden, R., The Rate of Hydrolysis of Phosphomonoester Dianions and the Exceptional Catalytic Proficiencies of Protein and Inositol Phosphatases, Proc. Natl. Acad. Sci. U. S. 100, 5607-5610, 2003.

Sievers, A., Beringer, M., Rodnina, M. V., Wolfenden, R., The Ribosome as an Entropy Trap, Proc. Natl. Acad. Sci. U. S. 101, 7897-7901, 2004.

Horvat, C. M., Wolfenden, R. V., A Persistent Pesticide Residue and the Unusual Catalytic Proficiency of a Dehalogenating Enzyme, Proc. Natl. Acad. Sci. U. S. 102, 16199-16202, 2005.

Schroeder, G. K., Lad, C., Wyman, P., Williams, N. H., Wolfenden, R. The Time Required for Water Attack at the Phosphorus Atom of Simple Phosphodiesters and of DNA, Proc. Natl. Acad. Sci. U. S. 103, 4052-4055, 2006.

Wolfenden, R., Degrees of Difficulty of Water-Consuming Reactions in the Absence of Enzymes, Chemical Reviews 106, 3379-3396, 2006.

Schroeder, G. K., Wolfenden, R., Rates of Spontaneous Disintegration of DNA, and the Catalytic Proficiencies of DNA Glycosylases and Deaminases, Biochemistry 46, 16368-16347, 2007.

Lewis, C. A., Jr., Wolfenden, UroD as a Benchmark for the Catalytic Proficiency of Enzymes, Proc. Natl. Acad. Sci. U. S. A. 105, 17328-17333, 2008.