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KMID : 0364819940320030222
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Korean Journal of Microbiology
1994 Volume.32 No. 3 p.222 ~ p.231
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Reaction Mechanism of Purine Nucleoside Phosphorylase and Effects of Reactive Agents for SH Group on the Enzyme in Saccharomyces cerevisiae
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최혜선:Choi Hye-Seon
저자없음:No authors listed
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Abstract
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Kinetic analysis was done to elucidatd the reaction mechanism of purine nucleoside phosphorylase (PNP) in Saccharomyces cerevisiae. The binary complexes of PNP·phosphate and PNP·ribose 1-phosphate were involved in the reaction mechanism. The initial velocity and product inhibition studies demonstrates were consistent with the predominant mechanism of the reaction being an ordered bi, bi reaction. The phosphate bound to the enzyme first, followed by nucleoside and base were the first product to leave, followed by ribose 1-phosphate. The kinetically suggested mechanism of PNP in S. cerevisiae was in agreement with the results of protection studies against the inactivation of the enzyme by sulfhydryl reagents, p-chloromercuribenzoate(PCMB) and 5,5’-dithiobisnitrobenzoate(DTNB). PNP was protected by ribose I-phosphate and phosphate, but not by nucleoside or base, supporting the reaction order of ordered bi, bi mechanism. PCMB or DTNB-inactivated PNP was totally reactivated by dithiothreitol(DTT) and the activity was returned to the level of 77% by 2-mercaptoethanol, indicating that inactivation was reversible. The kinetic behavior of the PCMB-inactivated enzyme had been change with higher K_m value of inosine and lower V_m, and was restored by DTT. Inactivation of enzyme by DTNB showed similar pattern of K_m value with that by PCMB, but had not changed the V_m value, significantly. Negative cooperativity was not found with PCMB or DTNB treated PNP at high concentration of phosphate.
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KEYWORD
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purine nucleoside phosphorylase, S. cerevisiae, reaction mechanism, PCMB, DTNB
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