The kinetic and mechanistic characterization of GAPDH and QPRTase from Mycobacterium tuberculosis

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.