title: Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase.
abstract: Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.
authors: Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461.
authors: Javier
authors: J
authors: Suarez
authors: Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461 vern.schramm@einstein.yu.edu.
authors: Vern L
authors: VL
authors: Schramm
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D002247:Carbon Isotopes / Q000737:chemistry; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium / Q000737:chemistry; D006151:Guanosine / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D009587:Nitrogen Isotopes / Q000737:chemistry; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012267:Ribosemonophosphates / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D007554:Isotopes; D009587:Nitrogen Isotopes; D012267:Ribosemonophosphates; D006151:Guanosine; C031154:ribose 1-phosphate; D006639:Histidine; D003903:Deuterium; D011683:Purine-Nucleoside Phosphorylase
keywords: Born–Oppenheimer enzymes; femtosecond dynamics; heavy enzymes; pre–steady-state chemistry; transition state coupling