Pre-steady-state transition-state analysis of the hydrolytic reaction catalyzed by purine nucleoside phosphorylase

P. C. Kline, Vern L. Schramm

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Abstract

The slow hydrolytic reaction catalyzed by calf spleen purine nucleoside phosphorylase [Kline, P. C., and Schramm, V. L. (1992) Biochemistry 31, 5964- 5973] has been investigated using pre-steady-state kinetic isotope effects and solvolysis studies. The stoichiometric reaction between enzyme and inosine forms 1 tool of free ribose per trimer of purine nucleoside phosphorylase and a tightly bound complex of enzyme and hypoxanthine. The experimental kinetic isotope effects from [1'-3H]-, [2'-3H]-, [4'-3H]-, [5'-3H]-, [1'-14C]-, and [9-15N]inosine are 1.151 ± 0.004, 1.145 ± 0.003, 1.006 ± 0.004, 1.028 ± 0.005, 1.045 ± 0.005, and 1.000 ± 0.005, respectively, for the pre-steady-state conditions. Substrate trapping experiments demonstrated that there is no detectable forward commitment to catalysis for inosine hydrolysis. In contrast, bound inosine is 2.1 times more likely to form product than to dissociate when the enzyme-inosine complex is exposed to saturating PO4. The lack of an observed 9-15N isotope effect is consistent with an internal equilibrium between enzyme- inosine and the enzyme-hypoxanthine-ribose complex in which N9 of hypoxanthine is protonated. The equilibrium occurs as a consequence of slow product release and tightly bound hypoxanthine (K(d) = 1.3 x 10-12 M). This internal equilibrium has a minimal effect on the intrinsic kinetic isotope effects from ribose since equilibrium isotope effects for conversion of inosine to ribose are near unity. When the single-turnover hydrolytic reaction was accomplished in 20% methanol, approximately 85% of the product sugar was 1-methylribose. Under these conditions, the anion-binding pocket fills with solvent which competes for the oxocarbenium ion of inosine formed at the transition state. In the presence of arsenate, no methanolysis of inosine occurs [Kline, P. C., and Schramm, V. L. (1993) Biochemistry 32, 13212-13219]. The results define a transition state with oxocarbenium ion character and weak participation of the attacking solvent nucleophile. Electrostatic potential surfaces of the transition states indicate that arsenate anion is more effective in neutralizing the oxocarbenium ion than is H2O.

Original languageEnglish (US)
Pages (from-to)1153-1162
Number of pages10
JournalBiochemistry
Volume34
Issue number4
StatePublished - 1995

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Purine-Nucleoside Phosphorylase
Inosine
Hypoxanthine
Isotopes
Ribose
Enzymes
Biochemistry
Ions
Kinetics
Anions
Nucleophiles
Static Electricity
Catalysis
Sugars
Methanol
Electrostatics
Hydrolysis
Spleen

ASJC Scopus subject areas

  • Biochemistry

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Pre-steady-state transition-state analysis of the hydrolytic reaction catalyzed by purine nucleoside phosphorylase. / Kline, P. C.; Schramm, Vern L.

In: Biochemistry, Vol. 34, No. 4, 1995, p. 1153-1162.

Research output: Contribution to journalArticle

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abstract = "The slow hydrolytic reaction catalyzed by calf spleen purine nucleoside phosphorylase [Kline, P. C., and Schramm, V. L. (1992) Biochemistry 31, 5964- 5973] has been investigated using pre-steady-state kinetic isotope effects and solvolysis studies. The stoichiometric reaction between enzyme and inosine forms 1 tool of free ribose per trimer of purine nucleoside phosphorylase and a tightly bound complex of enzyme and hypoxanthine. The experimental kinetic isotope effects from [1'-3H]-, [2'-3H]-, [4'-3H]-, [5'-3H]-, [1'-14C]-, and [9-15N]inosine are 1.151 ± 0.004, 1.145 ± 0.003, 1.006 ± 0.004, 1.028 ± 0.005, 1.045 ± 0.005, and 1.000 ± 0.005, respectively, for the pre-steady-state conditions. Substrate trapping experiments demonstrated that there is no detectable forward commitment to catalysis for inosine hydrolysis. In contrast, bound inosine is 2.1 times more likely to form product than to dissociate when the enzyme-inosine complex is exposed to saturating PO4. The lack of an observed 9-15N isotope effect is consistent with an internal equilibrium between enzyme- inosine and the enzyme-hypoxanthine-ribose complex in which N9 of hypoxanthine is protonated. The equilibrium occurs as a consequence of slow product release and tightly bound hypoxanthine (K(d) = 1.3 x 10-12 M). This internal equilibrium has a minimal effect on the intrinsic kinetic isotope effects from ribose since equilibrium isotope effects for conversion of inosine to ribose are near unity. When the single-turnover hydrolytic reaction was accomplished in 20{\%} methanol, approximately 85{\%} of the product sugar was 1-methylribose. Under these conditions, the anion-binding pocket fills with solvent which competes for the oxocarbenium ion of inosine formed at the transition state. In the presence of arsenate, no methanolysis of inosine occurs [Kline, P. C., and Schramm, V. L. (1993) Biochemistry 32, 13212-13219]. The results define a transition state with oxocarbenium ion character and weak participation of the attacking solvent nucleophile. Electrostatic potential surfaces of the transition states indicate that arsenate anion is more effective in neutralizing the oxocarbenium ion than is H2O.",
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N2 - The slow hydrolytic reaction catalyzed by calf spleen purine nucleoside phosphorylase [Kline, P. C., and Schramm, V. L. (1992) Biochemistry 31, 5964- 5973] has been investigated using pre-steady-state kinetic isotope effects and solvolysis studies. The stoichiometric reaction between enzyme and inosine forms 1 tool of free ribose per trimer of purine nucleoside phosphorylase and a tightly bound complex of enzyme and hypoxanthine. The experimental kinetic isotope effects from [1'-3H]-, [2'-3H]-, [4'-3H]-, [5'-3H]-, [1'-14C]-, and [9-15N]inosine are 1.151 ± 0.004, 1.145 ± 0.003, 1.006 ± 0.004, 1.028 ± 0.005, 1.045 ± 0.005, and 1.000 ± 0.005, respectively, for the pre-steady-state conditions. Substrate trapping experiments demonstrated that there is no detectable forward commitment to catalysis for inosine hydrolysis. In contrast, bound inosine is 2.1 times more likely to form product than to dissociate when the enzyme-inosine complex is exposed to saturating PO4. The lack of an observed 9-15N isotope effect is consistent with an internal equilibrium between enzyme- inosine and the enzyme-hypoxanthine-ribose complex in which N9 of hypoxanthine is protonated. The equilibrium occurs as a consequence of slow product release and tightly bound hypoxanthine (K(d) = 1.3 x 10-12 M). This internal equilibrium has a minimal effect on the intrinsic kinetic isotope effects from ribose since equilibrium isotope effects for conversion of inosine to ribose are near unity. When the single-turnover hydrolytic reaction was accomplished in 20% methanol, approximately 85% of the product sugar was 1-methylribose. Under these conditions, the anion-binding pocket fills with solvent which competes for the oxocarbenium ion of inosine formed at the transition state. In the presence of arsenate, no methanolysis of inosine occurs [Kline, P. C., and Schramm, V. L. (1993) Biochemistry 32, 13212-13219]. The results define a transition state with oxocarbenium ion character and weak participation of the attacking solvent nucleophile. Electrostatic potential surfaces of the transition states indicate that arsenate anion is more effective in neutralizing the oxocarbenium ion than is H2O.

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