TAILIEUCHUNG - Báo cáo khoa học: Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies Alteration of substrate specificity by replacement of Asn149 with Asp

To examine the interaction of human arginase II (EC ) with sub-strate 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. TheKmfor arginine (– mm) was not altered and the wild-type and mutant enzymes were essen-tially inactive on agmatine. | iFEBS Journal Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies Alteration of substrate specificity by replacement of Asn149 with Asp Vasthi Lopez Ricardo Alarcon Maria S. Orellana Paula Enriquez Elena Uribe Jose Martinez and Nelson Carvajal Departamento de Bioquimica y Biologia Molecular Facultad de Ciencias Biologicas Universidad de Concepcion Chile Keywords manganese ions histidine agmatinase activity arginase II human Correspondence N. Carvajal Departamento de Bioquimica y Biologia Molecular Facultad de Ciencias Biologicas Universidad de Concepcion Casilla 160-C Concepcion Chile Fax 56 41 239687 E-mail ncarvaja@ Received 24 May 2005 revised 13 July 2005 accepted 19 July 2005 doi To examine the interaction of human arginase II EC 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 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 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 Mn2 -subunit-1 whereas wild-type and His145Asn enzymes were half active and contained Mn2 -subunit-1 and Mn2 -subunit-1 respectively. Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with Kd of X 10-8 M for the wild-type and His145Asn enzymes and X 10-8 M for the His120Asn variant. Upon mutation the chromatographic behavior

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