Biochemical characterization of human GMP synthetase.
GMP synthetase (EC 6.3.5.2) plays a key role in the de novo synthesis of guanine nucleotides. It is a potential target for immunosuppressive therapy. Recently, the human enzyme was purified to homogeneity (Hirst, M., Haliday, E., Nakamura, J., and Lou, L. (1994) J. Biol. Chem. 269, 23830-23837). We now report the characterization of this enzyme in terms of its biochemical and kinetic properties. We found that there are distinct features of the human enzyme that has not been reported for GMP synthetase from other sources. There are two variant forms of human GMP synthetase. Their catalytic properties are very similar, although their isoelectric points are different. They most likely represent post-translational modification variants. Magnesium ion is required for enzyme activity, and the requirement is beyond levels needed for ATP chelation. Magnesium appears to be an essential activator and there may be more than one binding site. Interaction of GMP synthetase with xanthosine 5'-monophosphate (XMP), a substrate, exhibits sigmoidal kinetics with a Hill coefficient of 1.48 +/- 0.07. This positive cooperativity is not due to ligand-induced oligomerization, since GMP synthetase remains a monomer in the presence of XMP and other substrates. Decoyinine, a selective inhibitor of GMP synthetase, inhibits the human enzyme reversibly with uncompetitive inhibition kinetics toward glutamine and XMP and non-competitive kinetics toward ATP.
Nakamura
J
J
Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto, California 94304, USA.
Lou
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D011485:Protein Binding; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D019820:Xanthine
D007527:Isoenzymes; D012265:Ribonucleotides; D019820:Xanthine; C011141:xanthosine monophosphate; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing); D008274:Magnesium