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Title: The kinetic and mechanistic characterization of GAPDH and QPRTase from Mycobacterium tuberculosis
Authors: Wolfson-Stofko, Brett
Keywords: Biochemistry.
Issue Date: 2014
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 75-09(E), Section: B.;Advisors: John S. Blanchard.
Abstract: Mycobacterium tuberculosis (Mtb) is the etiological agent responsible for the disease tuberculosis (TB). Currently, Mtb has infected approximately one-third of the human population leading to nearly 1.3 million deaths worldwide in 2012. Of those infected, approximately 5-20% develop an active, symptomatic infection capable of spreading the bacterium. Due to increasing drug resistance, there is a desperate need for greater understanding of Mtb metabolism and the designing of novel anti-TB drugs.;Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD+) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Despite decades of research on GAPDH, none has been conducted on the GAPDH from Mycobacterium tuberculosis, a key component of glycolysis and cellular function. We report the first successful purification and mechanistic characterization of Mtb-GAPDH and found our data consistent with an unusual mechanism in which the free enzyme is really E-NAD+ and that NAD+ is required for Mtb-GAPDH stability. C158 was identified as the active site cysteine required for catalysis and H185 was shown to act as a catalytic base. Kinetic isotope effect studies suggest that the first-half reaction is rate-limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation.;Quinolinate phosphoribosyltransferase (QPRTase) is a type II phosphoribosyltransferase responsible for the conversion of quinolinate (QA) and phosphoribosyl pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN), pyrophosphate and carbon dioxide (CO2) in the de novo NAD+ biosynthesis pathway. Studies suggest that some pyrazinamide-resistant Mtb strains may be vulnerable to drugs disrupting de novo NAD+ biosynthesis. The formation of a proposed oxocarbenium-ion intermediate makes Mtb-QPRTase an attractive candidate for transition-state inhibitor design. The transition-state inhibitor GBE13-103a (TS) was screened against Mtb-QPRTase and was demonstrated to be ineffective. Efforts were made to phosphorylate TS (TSP) using adenosine kinase from Anopheles gambiae for screening against Mtb-QPRTase. TSP was identified using LC/MS but we were unable to purify significant amounts of TSP to screen against Mtb-QPRTase.
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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