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Neighboring group participation in the transition state of human purine nucleoside phosphorylase. 2007

Andrew S Murkin, and Matthew R Birck, and Agnes Rinaldo-Matthis, and Wuxian Shi, and Erika A Taylor, and Steven C Almo, and Vern L Schramm
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition-state analogues show His257 within hydrogen bonding distance of the 5'-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (Km/Ki) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His257 serves an important role in the early stages of transition-state formation. Whereas mutation of His257 resulted in little variation in the PNP x DADMe-ImmH x SO4 structures, His257Phe x ImmH x PO4 showed distortion at the 5'-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies of the remote 5'-(3)H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5'-(3)H intrinsic KIE to -3, -14, and 7%, respectively, while the BIEs contributed 2, 2, and -2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D011683 Purine-Nucleoside Phosphorylase An enzyme that catalyzes the reaction between a purine nucleoside and orthophosphate to form a free purine plus ribose-5-phosphate. EC 2.4.2.1. Inosine Phosphorylase,Nicotinamide Riboside Phosphorylase,Purine Nucleoside Phosphorylases,Nucleoside Phosphorylases, Purine,Phosphorylase, Inosine,Phosphorylase, Nicotinamide Riboside,Phosphorylase, Purine-Nucleoside,Phosphorylases, Purine Nucleoside,Purine Nucleoside Phosphorylase,Riboside Phosphorylase, Nicotinamide
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D016297 Mutagenesis, Site-Directed Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion. Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide
D018360 Crystallography, X-Ray The study of crystal structure using X-RAY DIFFRACTION techniques. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) X-Ray Crystallography,Crystallography, X Ray,Crystallography, Xray,X Ray Crystallography,Xray Crystallography,Crystallographies, X Ray,X Ray Crystallographies

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