title: A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase.
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(2+) 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-Mg(2+) 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-Mg(2+) 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.
authors: Laboratory for Macromolecular Analysis and Proteomics, Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors: F
authors: F
authors: Wang
authors: W
authors: W
authors: Shi
authors: E
authors: E
authors: Nieves
authors: R H
authors: RH
authors: Angeletti
authors: V L
authors: VL
authors: Schramm
authors: C
authors: C
authors: Grubmeyer
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium / Q000378:metabolism; D011756:Diphosphates / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D046911:Macromolecular Substances; D008274:Magnesium / Q000737:chemistry; D017616:Magnesium Compounds / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D011522:Protons; D011744:Pyrimidinones / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*
chemical_list: D011756:Diphosphates; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D017616:Magnesium Compounds; D010446:Peptide Fragments; D011522:Protons; D011744:Pyrimidinones; D011758:Pyrroles; C113100:immucillin G; D007532:Isoleucine; D010649:Phenylalanine; C034732:magnesium pyrophosphate; D003903:Deuterium; D007041:Hypoxanthine Phosphoribosyltransferase; D010434:Pepsin A; D007930:Leucine; D008274:Magnesium