Formation of a covalent Nε2-guanylylhistidyl reaction intermediate by the GTP:GTP guanylyltransferase from the brine shrimp Artemia

The chemical nature of the enzyme-nucleotide phosphoramidate reaction intermediate employed by the unique GTP:GTP guanylyltransferase from yolk platelets of Artemia franciscana cysts to synthesize diguanosine tetraphosphate (Gp(4)G) has been investigated. Labeling of the enzyme with [alpha-P-32]GTP followed by isolation of the labeled phosphoamino acid by periodate treatment and alkaline hydrolysis and comparison of the product with phosphoamino acid standards by thin-layer and ion-exchange chromatography showed that the linkage involves the N epsilon 2 ring nitrogen of an enzyme histidyl residue. Thus, this enzyme is distinct from the mRNA capping enzymes which can also synthesize Gp(4)G but which employ a lysyl-nucleotide intermediate. Based on its reaction mechanism and substrate specificity, GTP:GTP Guanylyltransferase may belong to the GAFH superfamily which includes the histidine triad proteins, Ap(4)A phosphorylases, and galactose-l-phosphate uridylyltransferase. (C) 1999 Academic Press.