A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase

F. Wang, W. Shi, E. Nieves, R. H. Angeletti, V. L. Schramm, C. Grubmeyer

Research output: Contribution to journalArticlepeer-review

35 Scopus citations


Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodium falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) exchange into amide bonds, quantitated by on-line HPLC and mass spectrometry, has been used to compare the dynamic and conformational properties of human HGPRT alone, the HGPRT·GMP·Mg complex, the HGPRT·IMP·MgPPi ↔ HGPRT·Hx·MgPRPP equilibrating mixture, and the transition-state analogue complex HGPRT·ImmGP·MgPPi. The rate and extent of H/D exchange of 26 peptic peptides, spanning 91% of the primary structure, have been monitored. Human HGPRT has 207 amide H/D exchange sites. After 1 h in D2O, HGPRT alone exchanges 160, HGPRT·GMP·Mg2+ exchanges 154, the equilibrium complex exchanges 139, and the transition-state analogue complex exchanges 126 of these amide protons. H/D exchange rates are correlated with structure for peptides in (1) catalytic site loops, (2) a connected peptide of the subunit interface of the tetramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HGPRT·GMP·Mg2+ and HGPRT·ImmGP·MgPPi complexes. Transition-state analogue binding strengthens the interaction between subunits and tightens the catalytic site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, and ligand exchange rates. Solvent exchange reveals loop dynamics in the free enzyme, Michaelis complexes, and the complex with the bound transition-state analogue. Proton transfer paths, rather than dynamic motion, are required to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.

Original languageEnglish (US)
Pages (from-to)8043-8054
Number of pages12
Issue number27
StatePublished - Jul 10 2001

ASJC Scopus subject areas

  • Biochemistry


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