Insights into the interaction of human arginase II with substrate and manganese ions by site‐directed mutagenesis and kinetic studies

摘要: To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, Hisl20Asn, Hisl45Asn Asnl49Asp mutations were introduced separately. About 53% 95% wild-type activitywere expressed by fully activated species Hisl20Asn variants, respectively. The K m for arginine (1.4-1.6 mM) was not altered mutant enzymes essentially inactive on agmatine. In contrast, almost undetectable activity arginine, but significant agmatinase (K = 2.5 ′ 0.2 markedly resistant to inhibition arginine. After dialysis against EDTA, variant totally in absence added Mn 2 + contained < 0.1 subunit - 1 , whereas half active 1.1 1.3 Manganese reactivation metal-free followed hyperbolic kinetics d 1.8 x 10 8 M 16.2 0.5 variant. Upon mutation, chromatographic behavior, tryptophan fluorescence properties (λ a 338-339 nm) sensitivity thermal inactivation altered. Asnl49→Asp mutation is proposed generate conformational change responsible specificity II. We also conclude that, contrast I, A more tightly bound metal ion