Purification and properties of GTP:GTP guanylyltransferase from encysted embryos of the brine shrimp Artemia.
GTP:GTP guanylyltransferase has been purified from the yolk platelets of Artemia cysts. The 480-kDa enzyme catalyzes the reversible reaction 2GTP<==>Gp4G + PPi and contains immunologically related polypeptides of 142, 88, and 45 kDa and a distinct 80-kDa component. The 88 and 45 kDa species can be covalently labeled with [alpha-32P]GTP. Even in crude extracts, the enzyme appears to be partially proteolyzed, suggesting that it is a nonfunctional residue of the pre-encystment stages of development. A native alpha 2 beta 2 structure comprising 2 mol each of the 142- and 80-kDa polypeptides is proposed. The reaction follows ping-pong kinetics with a covalent enzyme-guanylate intermediate containing a phosphoramidate linkage, probably phospholysine. The enzyme has two GTP-binding sites: a "donor" site in which the enzyme-guanylate is formed and which is highly specific for guanine nucleotides (GTP, p4G, dGTP, and GppNHp) and an "acceptor" site which additionally binds XTP, ITP, GDP, and ADP. Thus, the enzyme will form the homodinucleotides Gp4G, Gp5G, Gp3G, dGp4dG, and GppNHppG and the heterodinucleotides Gp4X, Gp4I, and Gp3A, but not Xp4X, Ip4I, or Gp4A. The Km for GTP was 6.7 mM and kcat was 1.6 s-1. XTP was a fully uncompetitive inhibitor of Gp4G synthesis while ITP was a partially uncompetitive inhibitor. In the reverse reaction, certain pyrophosphate analogs could substitute for PPi. The structure and mechanism of this enzyme suggest an evolutionary relationship to mRNA capping enzymes.
Liu
J J
JJ
Department of Biochemistry, University of Liverpool, United Kingdom.
McLennan
A G
AG
D000818:Animals; D001156:Artemia / Q000196:embryology / Q000201:enzymology*; D001665:Binding Sites; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D011756:Diphosphates / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000201:enzymology; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D013045:Species Specificity; D013379:Substrate Specificity
D011756:Diphosphates; D006150:Guanine Nucleotides; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; C020833:guanosinetriphosphate guanylyltransferase