title: The conserved serine-tyrosine dipeptide in Leishmania donovani hypoxanthine-guanine phosphoribosyltransferase is essential for catalytic activity.
abstract: 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 enzyme (Eads, J. C., Scapin, G. T., Xu, Y., Grubmeyer. C., and Sacchettini, 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 tetranitromethane or diethyl pyrocarbonate inactivated the enzyme completely, and peptide sequence analysis revealed that tetranitromethane treatment modified the Tyr residue within the Ser95-Tyr96 dipeptide. Analysis of site-directed mutants confirmed that both amino acids were vital for phosphoribosylation activity. Mutant HGPRTs, S95A, S95E, Y96F, and Y96V, exhibited dramatic reductions in their catalytic capabilities of 2-3 orders of magnitude, whereas HGPRTs containing conservative substitutions, S95C and S95T, displayed only a 2-3-fold decrease in kcat. Km values for the substrates of the forward and reverse reactions were largely unchanged for all HGPRT constructs, except for a 4-5-fold decrease in the Km value of the Y96F and Y96V mutants for phosphoribosylpyrophosphate. Expression of L. donovani hgprt constructs in Escherichia coli indicated that wild type and S95T HGPRTs complemented bacterial phosphoribosyltransferase deficiencies, whereas the S95A and S95C mutants complemented weakly, and the S95E, Y96F, and Y96V HGPRT did not support bacterial growth. These data authenticate that the Ser-Tyr dipeptide that is conserved among all members of the HGPRT family is essential for phosphoribosylation of purine nucleobases by HGPRT.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.
authors.forename: A
authors.initials: A
authors.lastname: Jardim
authors.forename: B
authors.initials: B
authors.lastname: Ullman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004151:Dipeptides / Q000737:chemistry*; D005816:Genetic Complementation Test; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000378:metabolism; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000494:pharmacology; D011487:Protein Conformation; D016415:Sequence Alignment; D012694:Serine; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine
chemical_list: D004151:Dipeptides; D007202:Indicators and Reagents; D010713:Phosphatidylcholines; D014443:Tyrosine; D012694:Serine; D007041:Hypoxanthine Phosphoribosyltransferase; D013774:Tetranitromethane; D004047:Diethyl Pyrocarbonate