Upon mutation of Asn130 to aspartate, the catalytic activity of human arginase I was reduced to ∼ 17% of wild‐type activity, the Km value for arginine was increased ∼ 9‐fold, and the kcat/Km value was reduced ∼ 50‐fold. The kinetic properties were much less affected by replacement of Asn130 with glutamine. In contrast with the wild‐type and N130Q enzymes, the N130D variant was active not only on arginine but also on its decarboxylated derivative, agmatine. Moreover, it exhibited no preferential substrate specificity for arginine over agmatine (kcat/Km values of 2.48 × 103 m−1·s−1 and 2.14 × 103 m−1·s−1, respectively). After dialysis against EDTA and assay in the absence of added Mn2+, the N130D mutant enzyme was inactive, whereas about 50% full activity was expressed by the wild‐type and N130Q variants. Mutations were not accompanied by changes in the tryptophan fluorescence properties, thermal stability or chromatographic behavior of the enzyme. An active site conformational change is proposed as an explanation for the altered substrate specificity and low catalytic efficiency of the N130D variant.

To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild‐type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The Km for arginine (1.4–1.6 mm) was not altered and the wild‐type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (Km = 2.5 ± 0.2 mm) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn2+ and contained < 0.1 Mn2+·subunit−1, whereas wild‐type and His145Asn enzymes were half active and contained 1.1 ± 0.1 Mn2+·subunit−1 and 1.3 ± 0.1 Mn2+·subunit−1, respectively. Manganese reactivation of metal‐free to half active species followed hyperbolic kinetics with Kd of 1.8 ± 0.2 × 10−8 m for the wild‐type and His145Asn enzymes and 16.2 ± 0.5 × 10−8 m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (λmax = 338–339 nm) and sensitivity to thermal inactivation were not altered. The Asn149→Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn2+A is the more tightly bound metal ion in arginase II.

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