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.
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 Zn(2+) led to, respectively, full and partial recovery of the enzyme activity. Excess Zn(2+) 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.
Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, 6681/92-A Av Ipiranga, 90619-900, Porto Alegre, RS, Brazil.
Jordana Dutra
JD
de Mendonça
Osao
O
Adachi
Leonardo Astolfi
LA
Rosado
Rodrigo Gay
RG
Ducati
Diogenes Santiago
DS
Santos
Luiz Augusto
LA
Basso
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism*; D002151:Calorimetry; D003035:Cobalt / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D015032:Zinc / Q000378:metabolism*
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D003035:Cobalt; C020001:3-dehydroquinate synthetase; D019761:Phosphorus-Oxygen Lyases; D015032:Zinc