Kinetics of Enzymatic Reactions
Princeton University, B. A., 1956; Oxford University B. A. (1958), M. A. (1960); Rockefeller Institute, Ph.D. (1964); NIH Research Career Development Award (1971-1976); Fellow, American Association for the Advancement of Science (1983); Member, Editorial Board, Bioorganic Chemistry (1983-); North Carolina Distinguished Chemist Award, ACS (1990);Member, Editorial Board, Bioorganic and Medicinal Chemistry and Bioorganic and Medicinal Chemistry Letters (1989-) Chairman, Biological Division, American Chemical Society (2000-); Elected, Member of National Academy of Sciences (2002); Elected, Fellow of American Academy of Arts and Sciences (2002)
Work in the Wolfenden Laboratory is animated by the idea -suggested by Michael Polanyi in 1921- that to enhance the rate of any reaction, a catalyst must bind the altered substrate very tightly in the transition state. In 1969, we showed that the increase in an enzyme's affinity for the substrate, in passing from the ground state to the transition state, matches or surpasses the factor by which the enzyme enhances the rate of a reaction. Accordingly, a stable molecule that resembles the altered substrate in the transition state should be a powerful inhibitor, much more tightly bound than the substrate in the ground state. Much of our research has focused on the quest for transition state analogue inhibitors. Earlier work supported by this grant led directly to the design of captopril, enalapril and lisinopril, the multisubstrate analogue ACE inhibitors that are now used to treat hypertension, and indirectly to transition state analogue retroviral protease inhibitors that are used to control HIV infection. One of our current projects is directed toward antagonists of antibiotic-resistant Pseudomonas aeruginosa, a bacterium that has been shown to play a major role in hospital infections. We are also working on the design of transition state analogue inhibitors of dihydroorotase, an enzyme responsible for the cyclization step in pyrimidine biosynthesis. This approach may lead to antimalarial drugs of simple structure that act selectively on the parasite which, unlike the human host, depends on this enzyme for its survival.
The second general goal of our work is to identify enzymes that produce large rate enhancements and offer particularly sensitive targets for drug design. Rate constants of reactions in the absence of enzymes furnish benchmarks for judging the proficiency of enzymes as catalysts. In addition they indicate the expected sensitivity to transition state analogue inhibitors. We are establishing these benchmarks by performing kinetic experiments in dilute aqueous solution at elevated temperatures, then using Eyring analysis to extrapolate rate constants to ordinary temperatures. In some cases, this requires temperatures that are relatively high. Using steel reaction bombs sealed with Teflon, one can conduct reactions at temperatures up to 270 ℃ where Teflon becomes amorphous. Steam pressure causes sealed quartz tubes to explode at roughly the same temperature. But when the sealed quartz tube is introduced into a steel pipe bomb along with some water outside the tube, the steam pressure becomes equalized, allowing experiments to be conducted at temperatures up to the critical point of water, 374 ℃. That innovation has recently allowed us conduct Arrhenius analysis of exceedingly slow reactions, with half-times exceeding the life of the earth, such as the hydrolysis of phosphate and sulfate esters.
Many textbooks carry the statement that enzymes enhance reaction rates "as much as 108-fold." In 1995, we reported that staphylococcal nuclease and orotidylate decarboxylase produced rate enhancements (kcat/knon) of ~1015 and ~1017, respectively. In 2003, we showed that phosphate monoestases produce rate enhancements of 1021. David Edwards in our laboratory showed in 2011 that S-O cleaving alkylsulfatases enhance the rate of hydrolysis of pentyl sulfate by a factor of ~1026. These comparisons allow a new appreciation of what evolution has accomplished, and a new perspective on the vast catalytic power that remains -for example- in a mutant enzyme whose activity is reduced to 1% of the wild type enzyme.
These experiments automatically generate information about a reaction's thermodynamics of activation, allowing those to be compared with the thermodynamics of activation of the enzyme reaction. Our experiments have shown that the catalytic effect of the peptidyltransferase center of the ribosome, and much of the catalytic effect of a group of kinases, is entropic in origin, consistent with an overwhelmingly important role for juxtaposition of the substrates in a position conducive to reaction. The role of the enzyme in most other reactions that we have examined is to lower the enthalpy of activation, consistent with an important role for H-bonds and other polar interactions with the enzyme in stabilizing the altered substrate in the transition state. Transition state analogue inhibitors should be designed to exploit these interactions.
These findings also have a startling bearing on the impact of temperature on the time required for the establishment of primordial biochemistry, and for the evolution of enzymes. In the absence of a catalyst, many biological reactions proceed with half-lives of hundreds, thousands or millions of years. Yet life appeared early during Earth's history. How did cellular chemistry -and the enzymes that make that chemistry possible- get started so quickly? We find that the hydrolysis of polysaccharides proceeds ~105-fold more rapidly when the temperature is raised from 25 to 100 ℃, while the rate of hydrolysis of phosphate monoester dianions increases ~107-fold. These temperature effects are most pronounced for the slowest reactions, collapsing the time that would have been required for slow reactions on a warm earth. Moreover, this behavior suggests a mechanism for enzyme evolution. A primitive catalyst, with modest activity at elevated temperatures, would have generated escalating rate enhancements as the surroundings cooled if, like modern enzymes, it catalyzed a reaction by reducing its heat of activation. Testing that possibility, we found that several simple catalysts, including PLP and metal ions, meet that criterion. Taken together, these findings greatly reduce the time that would have been required for early chemical evolution, countering the view that not enough time has passed for life to have evolved to its present level of complexity.
Edwards, D. R., Lohman, D. C., Wolfenden, R., Catalytic proficiency: the extreme case of S-O cleaving sulfatases, J. Am. Chem. Soc. 134, 525-531, 2012
Wolfenden, R., Benchmark reaction rates, the stability of biological molecules in water, and the evolution of catalytic power in enzymes, Ann. Rev. Biochem. 80, 645-667, 2011
Stockbridge, R. B.; Lewis, C. A., Jr.; Yuan, Y., Wolfenden, R., Impact of temperature on the time required for the establishment of primordial biochemistry, and for the evolution of enzymes, Proc. Nat. Acad Sci. U. S. A. 107, 22102-22105, 2010
Stockbridge, R.; Wolfenden, R., Phosphate monoester hydrolysis in cyclohexane, J. Am. Chem. Soc. 131, 18248-18249, 2009
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
Wolfenden, R., Degrees of Difficulty of Water-Consuming Reactions in the Absence of Enzymes, Chemical Reviews 106, 3379-3396, 2006
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
Callahan, B. P., Wolfenden, R., The Burden Borne by Urease, J. Am. Chem. Soc. 127, 10828-10829, 2005
Borchers, C. Marquez, V. E., Schroeder, G. K., Short, S. A., Snider, M. J., Speir, J. P., Wolfenden, R. Fourier, Transform Ion Cyclotron Resonance MS Reveals the Presence of a Water Molecule in an Enzyme Transition-State Analogue Complex, Proc. Natl. Acad. Sci. U. S. 101, 15341-15345, 2004
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