Phosphorylation of the antiviral precursor 9-(1,3-dihydroxy-2-propoxymethyl)guanine monophosphate by guanylate kinase isozymes.
Guanylate kinase was purified from human erythrocytes by affinity chromatography using GMP-agarose, and the four isozymes which are present were separated by chromatofocusing. The kinetic properties of each isozyme were analyzed with respect to the natural substrates GMP and dGMP, and the 5'-monophosphate derivatives of the antiviral nucleoside analogs 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and 9-(2-hydroxyethoxymethyl)guanine (ACV, Acyclovir). The analysis of substrate kinetics yielded Km values for DHPG 5'-monophosphate which were similar with all isozymes (42-54 microM), and about 3-fold higher than the Km values obtained for GMP. Km values obtained with ACV 5'-monophosphate were 10-20-fold higher than the GMP values and varied nearly 4-fold among isozymes (209-753 microM). GMP produced the highest enzyme velocities with all isozymes, followed by dGMP, DHPG 5'-monophosphate, and ACV 5'-monophosphate, in that order. Differences in maximal velocities among isozymes were generally small. DHPG 5'-monophosphate inhibited the isozymes by a simple competitive mechanism with respect to GMP. In contrast, ACV 5'-monophosphate acted as an apparent hyperbolic mixed-type inhibitor. Similar patterns of inhibition were obtained with all isozymes. It is probable that differences is the reactivity of DHPG 5'-monophosphate and ACV 5'-monophosphate with individual guanylate kinase isozymes do not contribute significantly to differences in their antiviral effects.
D000998:Antiviral Agents / Q000097:blood*; D004912:Erythrocytes / Q000201:enzymology*; D006150:Guanine Nucleotides / Q000097:blood*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives / Q000097:blood*; D051528:Guanylate Kinases; D006801:Humans; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D009703:Nucleoside-Phosphate Kinase / Q000097:blood* / Q000302:isolation & purification; D010766:Phosphorylation; D010770:Phosphotransferases / Q000097:blood*; D013379:Substrate Specificity
D000998:Antiviral Agents; D006150:Guanine Nucleotides; D007527:Isoenzymes; D006157:Guanosine Monophosphate; C043120:9-(1,3-dihydroxy-2-propoxymethyl)-GMP; D010770:Phosphotransferases; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases