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

F. Wang, W. Shi, Edward Nieves, R. H. Angeletti, Vern L. Schramm, C. Grubmeyer

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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
JournalBiochemistry
Volume40
Issue number27
DOIs
StatePublished - Jul 10 2001

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Hypoxanthine Phosphoribosyltransferase
Deuterium
Hydrogen
Peptides
Amides
Catalytic Domain
Proteins
Protons
Salvaging
Proton transfer
Enzymes
Mass spectrometry
Plasmodium falciparum
Structural properties
Hydrogen bonds
Malaria
Digestion
Mass Spectrometry
Ligands
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase. / Wang, F.; Shi, W.; Nieves, Edward; Angeletti, R. H.; Schramm, Vern L.; Grubmeyer, C.

In: Biochemistry, Vol. 40, No. 27, 10.07.2001, p. 8043-8054.

Research output: Contribution to journalArticle

Wang, F. ; Shi, W. ; Nieves, Edward ; Angeletti, R. H. ; Schramm, Vern L. ; Grubmeyer, C. / A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase. In: Biochemistry. 2001 ; Vol. 40, No. 27. pp. 8043-8054.
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abstract = "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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