The major emphasis during the next grant period will be on the use of 13C, 15N, and 18O isotope effects to determine the reaction mechanisms and/or obtain information on transition state structures of medically important enzymatic reactions. To complement the experimental studies of the transition state and catalytic mechanisms, computational studies will be carried out to help better characterize the atomic structure of the transition state. Computations will be carried out with two different approaches: active-site models studied with pure QM methods and complete enzyme models studied with QM/MM methods. 1. Sir2 is an important enzyme in cell signaling that transfers acetyl groups from acetylated lysine in proteins to the ribose of NAD+, which is cleaved into 2'-acetyl-ADP-ribose and nicotinamide. Isotope effects will be measured at C-1 of ribose and N-1 of nicotinamide in the NAD+ substrate, and at the carbonyl carbon and oxygen of the acetyl-peptide substrate in the reaction catalyzed by Sir2 to determine the reaction mechanism and distinguish between dissociative or associative transition states for the formation of the intermediate amidate. These kinetic isotope effects will also be measured in the presence of the Sir2 activators resveratrol and SRT1720 to determine if these compounds modify the enzyme's commitment to catalysis. 2. Polyamine Oxidase (PAO) is an amine oxidase that catalyzes the conversion of the acetylated polyamines, N1-acetylspermine and N1-acetylspermidine, back to their non-acetylated physiologically active forms. Present in all cells, polyamines are found in much higher levels in growing cells. Inhibitors of PAO would slow cell growth. 15N isotope effects will be measured for PAO with the slow substrate N,N'-dibenzyl-1,3- diaminopropane to distinguish the reaction mechanism (nucleophilic attack of amine on the flavin, a hydride transfer from the neutral amine, or a concerted proton transfer/amine addition). The results should direct the synthesis of substrate analogs that will inhibit the reactivation of acetyl polyamines in cancerous cells. 3. NAD+ synthetase (M. tuberculosis) is an important drug target for the treatment of tuberculosis. This is a multifunctional enzyme containing both a glutaminase and a synthetase domain. Isotope effects are currently being completed on this enzyme (M. tuberculosis) to elucidate the reaction mechanism and transition state structure.
This aim i ncludes computational methods and extends these 15N, 13C, and 18O isotope effect studies to the human NAD+ synthetase, which exhibits different kinetics than the microbial enzyme. Any difference in transition state structure between the enzymes from these two sources found experimentally through isotope effects and with current computational methods will aid the design of a potential inhibitor, which will be synthesized. The D497N and D497A mutant (M. tuberculosis) enzymes will be studied to investigate the possible role of this residue plays in proton transfer.

Public Health Relevance

This project is designed to use kinetic studies to determine enzyme mechanisms. The emphasis will be on the use of heavy atom isotope effects (carbon-13, nitrogen-15, and oxygen-18) along with deuterium ones and on computation of transition state structures.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-P (02))
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Anderson, Vernon
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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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
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
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
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
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
Poyner, Russell R; Anderson, Mark A; Bandarian, Vahe et al. (2006) Probing nitrogen-sensitive steps in the free-radical-mediated deamination of amino alcohols by ethanolamine ammonia-lyase. J Am Chem Soc 128:7120-1
Wright, S Kirk; DeClue, Michael S; Mandal, Ajay et al. (2005) Isotope effects on the enzymatic and nonenzymatic reactions of chorismate. J Am Chem Soc 127:12957-64

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