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

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodi um falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) excha nge into amide bonds, quantitated by on-line HPLC and mass spectrometry, ha s been used to compare the dynamic and conformational properties of human H GPRT alone, the HGPRT.GMP.Mg2+ complex, the HGPRT.IMP.MgPPi <----> HGPRT.Hx .MgPRPP equilibrating mixture, and the transition-state analogue complex HG PRT.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 ha s 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. HID exchange rates are correlated with structure for peptides in (1) catal ytic site loops, (2) a connected peptide of the subunit interface of the te tramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HG PRT.CMP.Mg2+ and HGPRT.ImmGP.MgPPi complexes. Transition-state analogue bin ding strengthens the interaction between subunits and tightens the catalyti c site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, a nd ligand exchange rates. Solvent exchange reveals loop dynamics in the fre e enzyme, Michaelis complexes, and the complex with the bound transition-st ate analogue. Proton transfer paths, rather than dynamic motion, are requir ed to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.