Catalytic and allosteric mechanism of AMP nucleosidase from primary, β-secondary, and multiple heavy atom kinetic isotope effects

David W. Parkin, Vern L. Schramm

Research output: Contribution to journalArticle

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Abstract

Adenosine 5′-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2′-2H]AMP (1.061 ± 0.002), [9-15N]AMP (1.030 ± 0.003), [1′-2H]AMP (1.045 ± 0.002), and [1′-14C]AMP (1.035 ± 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2′-2H]AMP effect (1.043 ± 0.002) and the [1′-2H]AMP effect (1.030 ± 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 ± 0.004 for [1′-2H,1′-14C]AMP and to 1.058 ± 0.002 for [9-15N,1′-14C]AMP with the enzyme in the absence of ATP. The primary 15N kinetic isotope effects were the same for acid- and enzyme-catalyzed hydrolysis while all of the secondary isotope effects were smaller for the enzyme-catalyzed hydrolysis. The secondary 3H isotope effects (with [1′-3H]AMP or [1-3H]ribose 5-phosphate) were approximately the same for the forward (1.047 ± 0.002) and reverse (1.06 ± 0.01) reactions, establishing an equilibrium isotope effect near unity and confirming the intrinsic nature of the isotope effects. The isotope effects indicate that the transition-state complex of AMP nucleosidase is oxycarbonium-like with hindered out-of-plane motion of the Cl′ hydrogen. In the presence of allosteric activator the bonding to Cl′ of ribose 5-phosphate in the transition state is unchanged, but the out-of-plane bending of the Cl′ and C2′ hydrogens is further restricted.

Original languageEnglish (US)
Pages (from-to)913-920
Number of pages8
JournalBiochemistry
Volume26
Issue number3
StatePublished - 1987
Externally publishedYes

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AMP nucleosidase
Isotopes
Atoms
Adenosine Monophosphate
Kinetics
Adenosine Triphosphate

ASJC Scopus subject areas

  • Biochemistry

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Catalytic and allosteric mechanism of AMP nucleosidase from primary, β-secondary, and multiple heavy atom kinetic isotope effects. / Parkin, David W.; Schramm, Vern L.

In: Biochemistry, Vol. 26, No. 3, 1987, p. 913-920.

Research output: Contribution to journalArticle

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AU - Parkin, David W.

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N2 - Adenosine 5′-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2′-2H]AMP (1.061 ± 0.002), [9-15N]AMP (1.030 ± 0.003), [1′-2H]AMP (1.045 ± 0.002), and [1′-14C]AMP (1.035 ± 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2′-2H]AMP effect (1.043 ± 0.002) and the [1′-2H]AMP effect (1.030 ± 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 ± 0.004 for [1′-2H,1′-14C]AMP and to 1.058 ± 0.002 for [9-15N,1′-14C]AMP with the enzyme in the absence of ATP. The primary 15N kinetic isotope effects were the same for acid- and enzyme-catalyzed hydrolysis while all of the secondary isotope effects were smaller for the enzyme-catalyzed hydrolysis. The secondary 3H isotope effects (with [1′-3H]AMP or [1-3H]ribose 5-phosphate) were approximately the same for the forward (1.047 ± 0.002) and reverse (1.06 ± 0.01) reactions, establishing an equilibrium isotope effect near unity and confirming the intrinsic nature of the isotope effects. The isotope effects indicate that the transition-state complex of AMP nucleosidase is oxycarbonium-like with hindered out-of-plane motion of the Cl′ hydrogen. In the presence of allosteric activator the bonding to Cl′ of ribose 5-phosphate in the transition state is unchanged, but the out-of-plane bending of the Cl′ and C2′ hydrogens is further restricted.

AB - Adenosine 5′-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2′-2H]AMP (1.061 ± 0.002), [9-15N]AMP (1.030 ± 0.003), [1′-2H]AMP (1.045 ± 0.002), and [1′-14C]AMP (1.035 ± 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2′-2H]AMP effect (1.043 ± 0.002) and the [1′-2H]AMP effect (1.030 ± 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 ± 0.004 for [1′-2H,1′-14C]AMP and to 1.058 ± 0.002 for [9-15N,1′-14C]AMP with the enzyme in the absence of ATP. The primary 15N kinetic isotope effects were the same for acid- and enzyme-catalyzed hydrolysis while all of the secondary isotope effects were smaller for the enzyme-catalyzed hydrolysis. The secondary 3H isotope effects (with [1′-3H]AMP or [1-3H]ribose 5-phosphate) were approximately the same for the forward (1.047 ± 0.002) and reverse (1.06 ± 0.01) reactions, establishing an equilibrium isotope effect near unity and confirming the intrinsic nature of the isotope effects. The isotope effects indicate that the transition-state complex of AMP nucleosidase is oxycarbonium-like with hindered out-of-plane motion of the Cl′ hydrogen. In the presence of allosteric activator the bonding to Cl′ of ribose 5-phosphate in the transition state is unchanged, but the out-of-plane bending of the Cl′ and C2′ hydrogens is further restricted.

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