Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity

Paul C. Kline, Vern L. Schramm

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Abstract

Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 × 10-4 s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 × 10-3 s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E·hypoxanthine complex dissociates at a rate of 1.9 × 10-6 s-1 at 4°C and 1.3 × 10-4 s-1 at 30°C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 × 10-12 M at 30°C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E·hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 μM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 μM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.

Original languageEnglish (US)
Pages (from-to)5964-5973
Number of pages10
JournalBiochemistry
Volume31
Issue number26
StatePublished - 1992

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Purine-Nucleoside Phosphorylase
Inosine
Hypoxanthine
Hydrolysis
Hypoxanthines
Phosphates
Enzymes
Ribose
Gel Chromatography
Conformations
Catalytic Domain
Gels
Guanosine
Guanine
Nucleosides
Stoichiometry

ASJC Scopus subject areas

  • Biochemistry

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Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity. / Kline, Paul C.; Schramm, Vern L.

In: Biochemistry, Vol. 31, No. 26, 1992, p. 5964-5973.

Research output: Contribution to journalArticle

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abstract = "Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 × 10-4 s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 × 10-3 s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E·hypoxanthine complex dissociates at a rate of 1.9 × 10-6 s-1 at 4°C and 1.3 × 10-4 s-1 at 30°C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 × 10-12 M at 30°C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E·hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 μM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 μM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.",
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N2 - Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 × 10-4 s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 × 10-3 s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E·hypoxanthine complex dissociates at a rate of 1.9 × 10-6 s-1 at 4°C and 1.3 × 10-4 s-1 at 30°C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 × 10-12 M at 30°C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E·hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 μM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 μM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.

AB - Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 × 10-4 s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 × 10-3 s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E·hypoxanthine complex dissociates at a rate of 1.9 × 10-6 s-1 at 4°C and 1.3 × 10-4 s-1 at 30°C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 × 10-12 M at 30°C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E·hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 μM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 μM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.

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