Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction

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Abstract

Purine nucleoside phosphorylase from calf spleen catalyzes the arsenolysis of inosine to form hypoxanthine and ribose 1-arsenate, which spontaneously hydrolyzes to ribose and arsenate. In the presence of H218O, no 18O is incorporated into ribose, demonstrating that ribose 1-arsenate hydrolysis occurs by attack of water on the arsenic atom. Rapid reaction kinetics at 20 °C result in a biphasic rate curve with the first turnover occurring at a rate of 20 s-1 followed by a steady-state rate of 2 s-1. The product burst is consistent with rapid steps for substrate binding and arsenolysis followed by rate-limiting hypoxanthine release at a rate of 2 s-1. Purine nucleoside phosphorylase with bound [14C] inosine was mixed with excess unlabeled inosine and arsenate to determine relative rates for reaction or dissociation of bound inosine. The commitment factor (product formed/inosine released) was 0.19 at saturating arsenate, indicating that inosine binds to free enzyme and that bound inosine is not in thermodynamic equilibrium with free substrate. At neutral pH, kinetic isotope effects for the phosphorolysis reaction are small, indicating kinetic suppression. Kinetic isotope effects for arsenolysis were measured with [1′-3H]-, [2′-3H]-, [1′-14C]-, [9-15N]-, [4′-3H]-, and [5′-3H]inosine to provide experimental values of 1.118 ± 0.003, 1.128 ± 0.003, 1.022 ± 0.005, 1.009 ± 0.004, 1.007 ± 0.003 and 1.028 ± 0.004 respectively. Following correction for commitment factors, the intrinsic isotope effects were matched to a geometric transition-state model selected by bond-energy bond order vibrational analysis. The transition state consistent with all isotope effects has a substantial decrease in the C1′-N9 glycosyl bond order, oxycarbonium character in the ribosyl ring, and weak participation of the arsenate nucleophile. Loss of the C1′-N9 bond is far ahead of the arsenate attack. The X-ray crystal structure for purine nucleoside phosphorylase with bound 9-deazainosine and inorganic sulfate places the nearest oxygen of the sulfate 4.2 Å from C1′ of the nucleoside analogue. This structure is consistent with a mechanism in which the ribosyl group is nearly dissociated from the base prior to attack of the arsenate.

Original languageEnglish (US)
Pages (from-to)13212-13219
Number of pages8
JournalBiochemistry®
Volume32
Issue number48
StatePublished - 1993

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Purine-Nucleoside Phosphorylase
Inosine
Ribose
Isotopes
Hypoxanthine
Sulfates
Kinetics
Intrinsic Factor
Nucleophiles
arsenic acid
Arsenic
Substrates
Thermodynamics
Nucleosides
Reaction kinetics
Hydrolysis
Spleen
Crystal structure
X-Rays
Oxygen

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction. / Schramm, Vern L.

In: Biochemistry®, Vol. 32, No. 48, 1993, p. 13212-13219.

Research output: Contribution to journalArticle

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abstract = "Purine nucleoside phosphorylase from calf spleen catalyzes the arsenolysis of inosine to form hypoxanthine and ribose 1-arsenate, which spontaneously hydrolyzes to ribose and arsenate. In the presence of H218O, no 18O is incorporated into ribose, demonstrating that ribose 1-arsenate hydrolysis occurs by attack of water on the arsenic atom. Rapid reaction kinetics at 20 °C result in a biphasic rate curve with the first turnover occurring at a rate of 20 s-1 followed by a steady-state rate of 2 s-1. The product burst is consistent with rapid steps for substrate binding and arsenolysis followed by rate-limiting hypoxanthine release at a rate of 2 s-1. Purine nucleoside phosphorylase with bound [14C] inosine was mixed with excess unlabeled inosine and arsenate to determine relative rates for reaction or dissociation of bound inosine. The commitment factor (product formed/inosine released) was 0.19 at saturating arsenate, indicating that inosine binds to free enzyme and that bound inosine is not in thermodynamic equilibrium with free substrate. At neutral pH, kinetic isotope effects for the phosphorolysis reaction are small, indicating kinetic suppression. Kinetic isotope effects for arsenolysis were measured with [1′-3H]-, [2′-3H]-, [1′-14C]-, [9-15N]-, [4′-3H]-, and [5′-3H]inosine to provide experimental values of 1.118 ± 0.003, 1.128 ± 0.003, 1.022 ± 0.005, 1.009 ± 0.004, 1.007 ± 0.003 and 1.028 ± 0.004 respectively. Following correction for commitment factors, the intrinsic isotope effects were matched to a geometric transition-state model selected by bond-energy bond order vibrational analysis. The transition state consistent with all isotope effects has a substantial decrease in the C1′-N9 glycosyl bond order, oxycarbonium character in the ribosyl ring, and weak participation of the arsenate nucleophile. Loss of the C1′-N9 bond is far ahead of the arsenate attack. The X-ray crystal structure for purine nucleoside phosphorylase with bound 9-deazainosine and inorganic sulfate places the nearest oxygen of the sulfate 4.2 {\AA} from C1′ of the nucleoside analogue. This structure is consistent with a mechanism in which the ribosyl group is nearly dissociated from the base prior to attack of the arsenate.",
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N2 - Purine nucleoside phosphorylase from calf spleen catalyzes the arsenolysis of inosine to form hypoxanthine and ribose 1-arsenate, which spontaneously hydrolyzes to ribose and arsenate. In the presence of H218O, no 18O is incorporated into ribose, demonstrating that ribose 1-arsenate hydrolysis occurs by attack of water on the arsenic atom. Rapid reaction kinetics at 20 °C result in a biphasic rate curve with the first turnover occurring at a rate of 20 s-1 followed by a steady-state rate of 2 s-1. The product burst is consistent with rapid steps for substrate binding and arsenolysis followed by rate-limiting hypoxanthine release at a rate of 2 s-1. Purine nucleoside phosphorylase with bound [14C] inosine was mixed with excess unlabeled inosine and arsenate to determine relative rates for reaction or dissociation of bound inosine. The commitment factor (product formed/inosine released) was 0.19 at saturating arsenate, indicating that inosine binds to free enzyme and that bound inosine is not in thermodynamic equilibrium with free substrate. At neutral pH, kinetic isotope effects for the phosphorolysis reaction are small, indicating kinetic suppression. Kinetic isotope effects for arsenolysis were measured with [1′-3H]-, [2′-3H]-, [1′-14C]-, [9-15N]-, [4′-3H]-, and [5′-3H]inosine to provide experimental values of 1.118 ± 0.003, 1.128 ± 0.003, 1.022 ± 0.005, 1.009 ± 0.004, 1.007 ± 0.003 and 1.028 ± 0.004 respectively. Following correction for commitment factors, the intrinsic isotope effects were matched to a geometric transition-state model selected by bond-energy bond order vibrational analysis. The transition state consistent with all isotope effects has a substantial decrease in the C1′-N9 glycosyl bond order, oxycarbonium character in the ribosyl ring, and weak participation of the arsenate nucleophile. Loss of the C1′-N9 bond is far ahead of the arsenate attack. The X-ray crystal structure for purine nucleoside phosphorylase with bound 9-deazainosine and inorganic sulfate places the nearest oxygen of the sulfate 4.2 Å from C1′ of the nucleoside analogue. This structure is consistent with a mechanism in which the ribosyl group is nearly dissociated from the base prior to attack of the arsenate.

AB - Purine nucleoside phosphorylase from calf spleen catalyzes the arsenolysis of inosine to form hypoxanthine and ribose 1-arsenate, which spontaneously hydrolyzes to ribose and arsenate. In the presence of H218O, no 18O is incorporated into ribose, demonstrating that ribose 1-arsenate hydrolysis occurs by attack of water on the arsenic atom. Rapid reaction kinetics at 20 °C result in a biphasic rate curve with the first turnover occurring at a rate of 20 s-1 followed by a steady-state rate of 2 s-1. The product burst is consistent with rapid steps for substrate binding and arsenolysis followed by rate-limiting hypoxanthine release at a rate of 2 s-1. Purine nucleoside phosphorylase with bound [14C] inosine was mixed with excess unlabeled inosine and arsenate to determine relative rates for reaction or dissociation of bound inosine. The commitment factor (product formed/inosine released) was 0.19 at saturating arsenate, indicating that inosine binds to free enzyme and that bound inosine is not in thermodynamic equilibrium with free substrate. At neutral pH, kinetic isotope effects for the phosphorolysis reaction are small, indicating kinetic suppression. Kinetic isotope effects for arsenolysis were measured with [1′-3H]-, [2′-3H]-, [1′-14C]-, [9-15N]-, [4′-3H]-, and [5′-3H]inosine to provide experimental values of 1.118 ± 0.003, 1.128 ± 0.003, 1.022 ± 0.005, 1.009 ± 0.004, 1.007 ± 0.003 and 1.028 ± 0.004 respectively. Following correction for commitment factors, the intrinsic isotope effects were matched to a geometric transition-state model selected by bond-energy bond order vibrational analysis. The transition state consistent with all isotope effects has a substantial decrease in the C1′-N9 glycosyl bond order, oxycarbonium character in the ribosyl ring, and weak participation of the arsenate nucleophile. Loss of the C1′-N9 bond is far ahead of the arsenate attack. The X-ray crystal structure for purine nucleoside phosphorylase with bound 9-deazainosine and inorganic sulfate places the nearest oxygen of the sulfate 4.2 Å from C1′ of the nucleoside analogue. This structure is consistent with a mechanism in which the ribosyl group is nearly dissociated from the base prior to attack of the arsenate.

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