THE CONSERVED SERINE-TYROSINE DIPEPTIDE IN LEISHMANIA-DONOVANI HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE IS ESSENTIAL FOR CATALYTIC ACTIVITY

Crystal structures of hypoxanthine-guanine phosphoribosyltransferase ( HGPRT) proteins have implied that the translocation of a flexible loop containing a highly conserved Ser-Tyr dipeptide is necessary for the protection of the proposed oxocarbonium ion transition state of the en zyme (Eads, J. C., Scapin, G. T., Xu, Y., Grubmeyer. C., and Sacchetti ni, J. C. (1994) Cell 78, 325-334; Schumacher, M. A., Carter, D., Roos , D. S., Ullman, B., and Brennan, R. G. (1996) Nature Struct. Biol. 3, 881-887). An essential role for this Ser-Tyr dyad in HGPRT catalysis has now been verified biochemically and genetically for the Leishmania donovani HGPRT employing a combination of protein modifying reagents and site-directed mutagenesis. Incubation of HGPRT with either tetrani tromethane or diethyl pyrocarbonate inactivated the enzyme completely, and peptide sequence analysis revealed that tetranitromethane treatme nt modified the Tyr residue within the Ser(95)-Tyr(96) dipeptide. Anal ysis of site-directed mutants confirmed that both amino acids were vit al for phosphoribosylation activity. Mutant HGPRTs, S95A, S95E, Y96F, and Y96V, exhibited dramatic reductions in their catalytic capabilitie s of 2-3 orders of magnitude, whereas HGPRTs containing conservative s ubstitutions, S95C and S95T, displayed only a 2-3-fold decrease in k(c at). K-m values for the substrates of the forward and reverse reaction s were largely unchanged for all HGPRT constructs, except for a 4-5-fo ld decrease in the K-m value of the Y96F and Y96V mutants for phosphor ibosylpyrophosphate. Expression of L. donovani hgprt constructs in Esc herichia cell indicated that wild type and S95T HGPRTs complemented ba cterial phosphoribosyltransferase deficiencies, whereas the S95A and S 95C mutants complemented weakly, and the S95E, Y96F, and Y96V HGPRT di d not support bacterial growth. These data authenticate that the Ser-T yr dipeptide that is conserved among all members of the HGPRT family i s essential for phosphoribosylation of purine nucleobases by HGPRT.