|
type |
Journal Article |
authors |
Alam M, Srivastava A, Dutta A, Sau AK |
title |
Biochemical and biophysical studies of Helicobacter pylori arginine decarboxylase, an enzyme important for acid adaptation in host |
journal |
IUBMB Life |
Activity |
4.1.1.19 |
Family |
4.1.1.19.1 |
sel |
selected |
ui |
29684243 |
year |
(2018) |
volume |
70 |
pages |
658-669 |
| |
keywords |
activity; circular dichroism; inhibition; thermal stability |
abstract |
Despite importance of arginine decarboxylase (ADC: EC 4.1.1.19) of Helicobacter pylori (H. pylori) 26695 pathogenic strain for acid adaptation in host, the enzyme has not yet been studied at a molecular level. Using combined approaches that include kinetic assays, site-directed mutagenesis, circular dichroism, heat-induced denaturation, analytical gel-filtration, and homology modeling, we report here a detailed investigation of H. pylori ADC. The pyridoxal 5'-phosphate (PLP)-dependent enzyme exhibits higher catalytic activity in the presence of Mg2+ ions at pH ∼8.5. Unlike other bacterial ADCs, this homolog exists as a hexamer. The higher thermal stability (Tm ∼65.8 ± 0.2 °C) of the enzyme observed from the heat-induced circular dichroism measurements indicates its secondary structural stabilization in the presence of PLP. The kinetic parameters Km and kcat of the enzyme are determined to be 3.4 ± 0.2 mM and 55.2 ± 1.0 min-1 , respectively. We elucidate that Cys487, a conserved residue located at the active-site, is involved in the catalysis, whose pKa value was estimated to be ∼7.2. The homology model of the protein show conserved α/β TIM barrel and β-sandwich domains, which are characteristic features of fold III decarboxylases. A lower sequence identity (∼21%) of this enzyme compared with its human counterpart has enabled us to screen putative inhibitors of H. pylori ADC. We found that α-difluoromethylarginine inhibits the activity of the H. pylori enzyme competitively with a Ki value ∼118 µM and thus it can serve as a basis to design inhibitors with higher efficacy against this ADC. |
last changed |
2018/10/05 11:38 |
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