TY - JOUR
T1 - A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase
AU - Wang, F.
AU - Shi, W.
AU - Nieves, E.
AU - Angeletti, R. H.
AU - Schramm, V. L.
AU - Grubmeyer, C.
PY - 2001/7/10
Y1 - 2001/7/10
N2 - 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·Mg2÷ 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.
AB - 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·Mg2÷ 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.
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U2 - 10.1021/bi010203f
DO - 10.1021/bi010203f
M3 - Article
C2 - 11434773
AN - SCOPUS:0035838469
SN - 0006-2960
VL - 40
SP - 8043
EP - 8054
JO - Biochemistry
JF - Biochemistry
IS - 27
ER -