Journal of Microbiology

Purification and characterization of purine nucleoside phosphorylase (PNP) in micrococcus luteus: Choi , Hye Seon; J. Microbiol. 1996;34(1):82-89.

Abstract: Purine nucleoside phosphorylase (PNP) was purified in Micrococcus luteus (M. luteus) using streptomycin sulfate and amomonium sulfate fractionation, three times by a Sephadex G-100 gel filtration and a DEAE-Sephadex A-50 ion exchange chromatography. The enzyme was purified 72 folds with a 11% recovery and showed a single band in a nondenaturing gel electrophoresis. The M. W. of PNP turned out to be 1.35 × 10^5 dalton in G-150 gel filtration chromatography. The stability of the enzyme was increased by treatment with both substrates, MgCI₂or CaCI₂, but not significantly kcal/mol. M. luteus PNP catalyzed the phosphorolysis of inosine, deoxyinosine, guanosine and deoxyguanosine with the Km value of 1.5 × 10^-3 M, 3.0 × 10^-3 M, 5.0 × 10^-4 M, respectively. The enzyme was reacted with adenosine, 1-methylnosine and 1-methylguanosine as substrates, which were shown to be poor substrates for mammalian enzyme.

Inhibition of purine nucleoside phosphorylase (PNP) in micrococcus luteus phenylglyoxal: Choi , Hye Seon; J. Microbiol. 1996;34(3):270-273.

Abstract: Micrococcus luteus purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been described previously. Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time-dependent manner by the arginine- specific modifying reagent phenylglyoxal. There was a linear relationship between the observed rate of inactivation and the phenylglyoxal concentration. At 30℃ the bimolecular rate constant for the modification was 0.015 min^-1 mM^-1 in 50 mM NaHCO₃buffer, pH 7.5. The plot of logk versus log phenylglyoxal concentration was a strainght line with a slope enzyme. Preincuation with saturated solutions of substrates protected the enzyme from inhibition of phenylglyoxal, indicating that reactions with phenylglyoxal were directed at arginyl residues essential for the catalytic functioning of the enzyme.

Catalytic mechanism and inhibition studies of purine nucleoside phosphorylase (PNP) in micrococcus luteus: Choi , Hye Seon; J. Microbiol. 1997;35(1):15-20.

Abstract: Kinetic studies were done to elucidate the reaction mechanism of purine nucleoside phosphorylase (PNP) in Micrococcus Luteus. PNP catalyzes the reversible phosphorolysis of ribonucleosides to their respective base. The effect of alternative competing substrates suggested that a single enzyme was involved in binding to the active site for all purine nucleosides, inosine, deoxyiosine, guanosine, deoxyguanosine, adenosine and deoxyadenosine. Affinity studies showed that pentose moiety reduced the binding capacity and methylation of ring N-1 of inosine and guanosine had little effect on binding to bacterial enzyme, whereas these compounds did not bind to the mammalian enzymes. The initial velocity and product inhibition studies demonstrated that the predominant mechanism of reaction was an ordered bi, bi reaction. The nucleoside bound to the enzyme first, followed by phosphate. Ribose 1-phosphate was the first product to leave, followed by base.

Chemical Midification of Purin Nucleoside Phosphorulase in Serratia marcescens: Choi , Hye Seon; J. Microbiol. 1998;36(2):74-79.

Abstract: Serratia marcescens purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been previously described(Choi, H.S. 1998. Biosci. Biotechnol. Biochem. 62, 667-671). Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time dependent manner by phenylglyoxal or diethylpyrocarbonate (DEPC). There was a linear relationship between the observed rate of inactivation and the phenylglyoxal or DEPC concentration. At 30℃ the bimolecular rate constant for the modification was 0.22 mM^-1 min^-1 in 50 mM NaHCO_3 buffer, pH 7.5, for phenylglyoxal and 1.33 mM^-1min^-1 in 50 mM sodium cotrate, pH 6.0, for DEPC. Preincubation with saturated solutions of substrates protected the enzyme from inhibition by kphenylglyoxal and DEPC, indicating that reactions with these reagents were directed at arginyl and histidyl residues, respectively, which are essential for the catalytic function of the enzyme.

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