title: Polyphosphate kinase from M. tuberculosis: an interconnect between the genetic and biochemical role.
abstract: The enzyme Polyphosphate Kinase (PPK) catalyses the reversible transfer of the terminal γ-Pi of ATP to form a long chain Polyphosphate (PolyP). Using an IPTG inducible mycobacterial vector, the vulnerability of this gene has been evaluated by antisense knockdown experiments in M. tuberculosis. Expression profiling studies point to the fact that down regulation of PPK caused cidality during the late phase in contrast to its bacteriostatic mode immediately following antisense expression. PPK thus seems to be a suitable anti-tubercular drug target. The enzyme which is a tetramer has been cloned in E. coli and purified to homogeneity. An enzyme assay suitable for High Throughput Screening was optimized by using the statistical Taguchi protocol and the kinetic parameters determined. The enzyme displayed a strong product inhibition by ADP. In order to accurately estimate the product inhibition, progress curve analysis of the enzyme reaction was monitored. The kinetic equation describing the progress curve was suitably modified by taking into account the product inhibition. The reversible nature of the enzyme indicated a possibility of a two way ATP↔ADP switch operating in the bacteria as a response to its growth requirement.
authors.affiliation: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors.forename: Vijayalakshmi
authors.initials: V
authors.lastname: Jagannathan
authors.forename: Parvinder
authors.initials: P
authors.lastname: Kaur
authors.forename: Santanu
authors.initials: S
authors.lastname: Datta
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000995:Antitubercular Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D015536:Down-Regulation; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008957:Models, Genetic; D015233:Models, Statistical; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*; D014048:Tolonium Chloride / Q000494:pharmacology
chemical_list: D000995:Antitubercular Agents; D014048:Tolonium Chloride; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase