The purpose of this project is to develop kinetic tools for studying enzyme mechanisms, and to apply them to representative enzymes. During the coming grant period the emphasis will be on the use of (13)C, (15)N, and (18)0 isotope effects: 1) Isotope effects in the beta-lambda bridge and lambda bridge and lambda-nonbridge oxygens of ATP and several analogs will be used to study the reactions of myosin ATPase and several kinases. (18)0 isotope effects will be used to define the transition state structure of kanamycin nucleotidyl transferase. Triesters containing a cyclic ethylene group and a leaving group with pK 8.6 will permit observation of isotope effects that accompany phosphorane formation and pseudorotation during hydrolysis. 2) With human malic enzyme, (13)C isotope effects will be measured at C-4 of malate or 2-deuteromalate with NAD or acetylpyridine-NAD to pin down the mechanism and rate limiting steps. 3) L-ribulokinase will be crystallized with AMPPNP and either D- or L-ribulose in the active site and the structures determined by X-ray. 4) The compound giving the upfield (31)P NMR signal that may represent carboxy-P in solutions of phosphate in DMF or DMSO containing CO2 will be characterized and tested as a substrate for appropriate enzymes. 5) The mechanism of asparagine synthetase will be studied with (13)C, (15)N and (18)O isotope effects. 6) The mechanism of isocitrate dehydrogenase will be studied with (13)C and deuterium isotope effects to determine the rate limiting steps with alternate substrates and mutant enzymes. 7) The (13)C isotope effect in the =CH2 group of chorismate will be determined to characterize the transition state for the chorismate mutase reaction. 8) (13)C and (15)N isotope effects will be used to study the mechanism of ornithine transcarboxylase. 9) Other collaborations will determine isotope effects on cytidine deaminase, non-enzymatic decarboxylation of hydroxybenzoates, and on selected fiavoprotein-catalyzed reactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM018938-31
Application #
6489909
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
1977-01-01
Project End
2004-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
31
Fiscal Year
2002
Total Cost
$300,540
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Reinhardt, Laurie A; Thoden, James B; Peters, Greg S et al. (2013) pH-rate profiles support a general base mechanism for galactokinase (Lactococcus lactis). FEBS Lett 587:2876-81
Smith, Brian C; Anderson, Mark A; Hoadley, Kelly A et al. (2012) Structural and kinetic isotope effect studies of nicotinamidase (Pnc1) from Saccharomyces cerevisiae. Biochemistry 51:243-56
Thoden, James B; Reinhardt, Laurie A; Cook, Paul D et al. (2012) Catalytic mechanism of perosamine N-acetyltransferase revealed by high-resolution X-ray crystallographic studies and kinetic analyses. Biochemistry 51:3433-44
Saylor, Benjamin T; Reinhardt, Laurie A; Lu, Zhibing et al. (2012) A structural element that facilitates proton-coupled electron transfer in oxalate decarboxylase. Biochemistry 51:2911-20
Marlier, John F; Robins, Lori I; Tucker, Kathryn A et al. (2010) A kinetic and isotope effect investigation of the urease-catalyzed hydrolysis of hydroxyurea. Biochemistry 49:8213-9
Van Vleet, Jeremy; Kleeb, Andreas; Kast, Peter et al. (2010) 13C isotope effect on the reaction catalyzed by prephenate dehydratase. Biochim Biophys Acta 1804:752-4
Pinto-Tomas, Adrian A; Anderson, Mark A; Suen, Garret et al. (2009) Symbiotic nitrogen fixation in the fungus gardens of leaf-cutter ants. Science 326:1120-3
Marlier, John F; Fogle, Emily J; Cleland, W W (2008) A heavy-atom isotope effect and kinetic investigation of the hydrolysis of semicarbazide by urease from jack bean (Canavalia ensiformis). Biochemistry 47:11158-63
Van Vleet, Jeremy L; Reinhardt, Laurie A; Miller, Brian G et al. (2008) Carbon isotope effect study on orotidine 5'-monophosphate decarboxylase: support for an anionic intermediate. Biochemistry 47:798-803
Ralph, Erik C; Hirschi, Jennifer S; Anderson, Mark A et al. (2007) Insights into the mechanism of flavoprotein-catalyzed amine oxidation from nitrogen isotope effects on the reaction of N-methyltryptophan oxidase. Biochemistry 46:7655-64

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