TY - JOUR
T1 - Kinetic mechanism determination and analysis of metal requirement of dehydroquinate synthase from Mycobacterium tuberculosis H37Rv
T2 - An essential step in the function-based rational design of anti-TB drugs
AU - De Mendonça, Jordana Dutra
AU - Adachi, Osao
AU - Rosado, Leonardo Astolfi
AU - Ducati, Rodrigo Gay
AU - Santos, Diogenes Santiago
AU - Basso, Luiz Augusto
PY - 2011/1/1
Y1 - 2011/1/1
N2 - 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.
AB - 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.
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U2 - 10.1039/c0mb00085j
DO - 10.1039/c0mb00085j
M3 - Article
C2 - 20978656
AN - SCOPUS:78650153694
SN - 1742-206X
VL - 7
SP - 119
EP - 128
JO - Molecular BioSystems
JF - Molecular BioSystems
IS - 1
ER -