The theory of absolute reaction rates implies that the function of a catalyst depends on its affinity for activated intermediates in substrate transformation, in preference to the substrate in the ground state. These two forms of the substrate, which differ only slightly in structure, differ in binding affinity for an enzyme's active site by factors that equal or exceed the rate enhancement that an enzyme produces. Enzymes are extremely effective catalysts, enhancing rates of reaction of natural substrates by factors as large as 1017. Accordingly, if a stable compound could be made to resemble an activated intermediate in substrate transformation, then such an analog should be bound very much more tightly than the substrate itself. Through structural studies of enzyme complexes with transition state analogs and multisubstrate analogs, we are trying to gain a better understanding of the forces that re responsible for an enzyme's ability to discriminate between compounds that resemble each other so closely as the substrate in the ground state and the transition state. During the most recent project period, we have observed very high levels of structural discrimination between compounds that differ only in variations in ligand structure will be used, along with studies of enzyme-inhibitor complexes by exact physical methods, to analyze the extremely strong interactions that appear to be responsible for the binding by cytidine deaminase and adenosine deaminase of analogs of covalently hydrated intermediates in substrate transformation. We will explore the binding specificity and mechanism of action of glycosyl-transferring enzymes including beta- glucosidase, beta-galactosidase and nucleoside deoxyribosyltransferase, to gain a better understanding of the contributions of individual substituents to binding affinity. The binding properties of prolidase, a manganese- dependent peptidase that is extraordinarily sensitive to inhibition by polybasic acids including the natural product P-enolpyruvate, will be investigated. Other targets for inhibitor design will include several enzymes involved in the metabolism of purines: adenylosuccinate synthetase, IMP dehydrogenase and IMP cyclohydrolase.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
Special Emphasis Panel (NSS)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
Zip Code
Lewis Jr, Charles A; Shen, Lin; Yang, Weitao et al. (2017) Three Pyrimidine Decarboxylations in the Absence of a Catalyst. Biochemistry 56:1498-1503
Lewis Jr, Charles A; Crayle, Jesse; Zhou, Shuntai et al. (2016) Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history. Proc Natl Acad Sci U S A 113:8194-9
Carter Jr, Charles W; Wolfenden, Richard (2016) tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry. RNA Biol 13:145-51
Wolfenden, Richard; Lewis Jr, Charles A; Yuan, Yang et al. (2015) Temperature dependence of amino acid hydrophobicities. Proc Natl Acad Sci U S A 112:7484-8
Carter Jr, Charles W; Wolfenden, Richard (2015) tRNA acceptor stem and anticodon bases form independent codes related to protein folding. Proc Natl Acad Sci U S A 112:7489-94
Wolfenden, Richard (2014) Massive thermal acceleration of the emergence of primordial chemistry, the incidence of spontaneous mutation, and the evolution of enzymes. J Biol Chem 289:30198-204
Lewis Jr, Charles A; Wolfenden, Richard (2014) The nonenzymatic decomposition of guanidines and amidines. J Am Chem Soc 136:130-6
Zhou, Xin; Chou, Tsui-Fen; Aubol, Brandon E et al. (2013) Kinetic mechanism of human histidine triad nucleotide binding protein 1. Biochemistry 52:3588-600
Lohman, Danielle C; Edwards, David R; Wolfenden, Richard (2013) Catalysis by desolvation: the catalytic prowess of SAM-dependent halide-alkylating enzymes. J Am Chem Soc 135:14473-5
Schroeder, Gottfried K; Zhou, Li; Snider, Mark J et al. (2012) Flight of a cytidine deaminase complex with an imperfect transition state analogue inhibitor: mass spectrometric evidence for the presence of a trapped water molecule. Biochemistry 51:6476-86

Showing the most recent 10 out of 26 publications