Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding.

Published

Journal Article

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.

Full Text

Duke Authors

Cited Authors

  • Balendiran, GK; Molina, JA; Xu, Y; Torres-Martinez, J; Stevens, R; Focia, PJ; Eakin, AE; Sacchettini, JC; Craig, SP

Published Date

  • May 1999

Published In

Volume / Issue

  • 8 / 5

Start / End Page

  • 1023 - 1031

PubMed ID

  • 10338013

Pubmed Central ID

  • 10338013

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1110/ps.8.5.1023

Language

  • eng

Conference Location

  • United States