Mutational analysis of substrate recognition by human arginase type I--agmatinase activity of the N130D variant.
Upon mutation of Asn130 to aspartate, the catalytic activity of human arginase I was reduced to approximately 17% of wild-type activity, the Km value for arginine was increased approximately 9-fold, and the kcat/Km value was reduced approximately 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 x 10(3) M(-1) x s(-1) and 2.14 x 10(3) M(-1) x 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.
Alarcón
Ricardo
R
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Chile.
Orellana
María S
MS
Neira
Benita
B
Uribe
Elena
E
García
José R
JR
Carvajal
Nelson
N
D019943:Amino Acid Substitution; D001119:Arginase / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature; D014512:Ureohydrolases / Q000235:genetics* / Q000378:metabolism*
D014512:Ureohydrolases; D001119:Arginase; C019625:agmatinase