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J Bioenerg Biomembr ; 48(6): 557-567, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27683242

RESUMO

Mycobacterium tuberculosis represents one of the world's most devastating infectious agents - with one third of the world's population infected and 1.5 million people dying each year from this deadly pathogen. As part of an effort to identify targets for therapeutic intervention, we carried out the kinetic characterization of the product of gene rv1700 of M. tuberculosis. Based on its sequence and its structure, the protein had been tentatively identified as a pyrophosphohydrolase specific for adenosine diphosphate ribose (ADPR), a compound involved in various pathways including oxidative stress response and tellurite resistance. In this work we carry out a kinetic, mutational and structural investigation of the enzyme, which provides a full characterization of this Mt-ADPRase. Optimal catalytic rates were achieved at alkaline pH (7.5-8.5) with either 0.5-1 mM Mg2+ or 0.02-1 mM Mn2+. K m and k cat values for hydrolysis of ADPR with Mg2+ ions are 200 ± 19 µM and 14.4 ± 0.4 s-1, and with Mn2+ ions are 554 ± 64 µM and 28.9 ± 1.4 s-1. Four residues proposed to be important in the catalytic mechanism of the enzyme were individually mutated and the kinetics of the mutant enzymes were characterized. In the four cases, the K m increased only slightly (2- to 3-fold) but the k cat decreased significantly (300- to 1900-fold), confirming the participation of these residues in catalysis. An analysis of the sequence and structure conservation patterns in Nudix ADPRases permits an unambiguous identification of members of the family and provides insight into residues involved in catalysis and their participation in substrate recognition in the Mt-ADPRase.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Mycobacterium tuberculosis/enzimologia , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Hidrolases/metabolismo , Cinética , Mutação , Mycobacterium tuberculosis/genética , Pirofosfatases/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
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