Phosphorylation of acyclovir (acycloguanosine) monophosphate by GMP kinase.

The levels of acyclo-GMP (9([2-(phosphonooxy)ethoxy]methyl)guanine) phosphorylating activity were not significantly different in uninfected and herpes implex virus-infected Vero cells. The relative rates of phosphorylation of acyclo-GMP and GMP remained unchanged after gel filtration where both activities were observed to co-elute at a volume corresponding to a molecular weight of 22,000. This suggested that host cell GMP kinase and not a virally induced enzyme catalyzed the phosphorylation of acyclo-GMP. In order to further substantiate this, human erythrocyte GMP kinase was purified 31,000-fold by affinity chromatography. It catalyzed the phosphorylation of acyclo-GMP and GMP with the same velocity ratio as was observed in crude extracts of both erythrocytes and Vero cells. With the purified enzyme, acyclo-GMP had an apparent K,,, of 0.33 m and an apparent V,, of 3% that of GMP. Common product analysis of the two alternative substrates, 6-thioguanosine-B'-P and acyclo-GMP, indicated that a single enzyme was responsible for the phosphorylation of both compounds. As an alternative substrate inhibitor, acyclo-GMP exhibited hyperbolic mixed type inhibition (apparent Ki = 0.24 nm) relative to GMP. In contrast, 6-thioguanosine-5'-P displayed competitive alternative substrate inhibition against both GMP and acyclo-GMP with Ki values of 3.0 n m and 2.4 r n ? respectively. Of the nucleotide kinases tested (AMP kinase, GMP kinase, nucleoside monophosphate kinase from beef liver, T4-induced deoxynucleotide kinase, and herpes simplex virus-induced thymidylate kinase), only GMP kinase phosphorylated acyclo-GMP.