Kinetic mechanism determination and analysis of metal requirement of dehydroquinate synthase from Mycobacterium tuberculosis H37Rv: An essential step in the function-based rational design of anti-TB drugs

Jordana Dutra De Mendonça, Osao Adachi, Leonardo Astolfi Rosado, Rodrigo Gay Ducati, Diogenes Santiago Santos, Luiz Augusto Basso

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

The number of new cases of tuberculosis (TB) arising each year is increasing globally. Migration, socio-economic deprivation, HIV co-infection and the emergence of drug-resistant strains of Mycobacterium tuberculosis, the main causative agent of TB in humans, have all contributed to the increasing number of TB cases worldwide. Proteins that are essential to the pathogen survival and absent in the host, such as enzymes of the shikimate pathway, are attractive targets to the development of new anti-TB drugs. Here we describe the metal requirement and kinetic mechanism determination of M. tuberculosis dehydroquinate synthase (MtDHQS). True steady-state kinetic parameters determination and ligand binding data suggested that the MtDHQS-catalyzed chemical reaction follows a rapid-equilibrium random mechanism. Treatment with EDTA abolished completely the activity of MtDHQS, and addition of Co 2+ and Zn2+ led to, respectively, full and partial recovery of the enzyme activity. Excess Zn2+ inhibited the MtDHQS activity, and isotitration microcalorimetry data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. We also report measurements of metal concentrations by inductively coupled plasma atomic emission spectrometry. The constants of the cyclic reduction and oxidation of NAD+ and NADH, respectively, during the reaction of MtDHQS was monitored by a stopped-flow instrument, under single-turnover experimental conditions. These results provide a better understanding of the mode of action of MtDHQS that should be useful to guide the rational (function-based) design of inhibitors of this enzyme that can be further evaluated as anti-TB drugs.

Original languageEnglish (US)
Pages (from-to)119-128
Number of pages10
JournalMolecular BioSystems
Volume7
Issue number1
DOIs
StatePublished - Jan 1 2011

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Kinetic mechanism determination and analysis of metal requirement of dehydroquinate synthase from Mycobacterium tuberculosis H37Rv: An essential step in the function-based rational design of anti-TB drugs'. Together they form a unique fingerprint.

  • Cite this