Purification and Properties of Guanidinoacetate Kinase from a Polychaete,Perinereissp.

Abstract

Guanidinoacetate kinase (ATP: guanidinoacetate N-phosphotransferase, EC 2.7.3.1) was extracted and purified to homogeneity from a polychaete, Perinereis sp., by ammonium sulfate fractionation and chromatographies with Butyl-Toyopearl 650C, Sephadex G-200, and DEAESephacel gels. Perinereis guanidinoacetate kinase was separated into two active fractions (I and II) by DEAE-Sephacel column chromatography, which had similar specific activities, and so some properties of guanidinoacetate kinase II (a major fraction) were investigated. The molecular weight of the enzyme was 90,000 on gel filtration and it was composed of two non-identical subunits (47,000 and 45,000). The enzyme showed optimum activity around pH 8.1 and was stable from pH 5.5 to 9.5 in the forward reaction. It was strictly specific for guanidinoacetate, and ATP was the most effective phosphoryl group donor for the forward reaction. The enzyme was activated by metal ions such as Mg2 + or Mn 2 + while Ca 2 + was 29% as active as Mg2 + • The Km values were 4.1 mM and 0.80 mM for guanidinoacetate and ATP, respectively. The enzyme activity was strongly inhibited by sulfhydryl-group-blocking agents, Hg2+, N-bromosuccinimide, and SDS. ADP and agmatine were competitive inhibitors.