Effects of allosteric activation on the primary and secondary kinetic isotope effects for three AMP nucleosidases

D. W. Parkin, Vern L. Schramm

Research output: Contribution to journalArticle

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Abstract

Kinetic isotope effects (V/K) were measured with AMP nucleosidases isolated from Azotobacter vinelandii, from a V(max) mutant enzyme of A. vinelandii and from Escherichia coli. Specifically labeled AMP substrates were used to measure 3H secondary and 14C primary kinetic isotope effects on the N-glycosidic bond hydrolysis of AMP in the presence and absence of the allosteric activator, MgATP. Use of the three enzymes, variable MgATP concentration, a poor substrate (dAMP), and variable pH has allowed determination of the isotope effects over a 5000-fold range in the catalytic turnover number. The primary kinetic isotope effects were 1.025 ± 0.004 and 1.041 ± 0.006 for the native A. vindelandii enzyme and mutant enzyme, respectively, and were independent of MgATP concentration. The E. coli AMP nucleosidase had a primary isotope effect of 1.019 ± 0.003 which was also independent of MgATP concentration. The secondary kinetic isotope effect decreased from 1.066 ± 0.003 to 1.045 ± 0.002 for the native enzyme from A. vinelandii as the concentration of MgATP increased from 0 to 500 μM. The secondary isotope effect of the mutant ezyme remained constant at 1.088 ± 0.005 as the MgATP concentration increased from 0 to 500 μM. The secondary isotope effect of the E. coli enzyme showed a similar pattern to that of the native enzyme, decreasing from 1.087 ± 0.003 to 1.050 ± 0.003 as the enzyme was saturated with MgATP at a constant concentration of AMP. Saturation with AMP in the absence of MgATP gave similar results and suggested that AMP can cause the allosteric transition. Both the primary and secondary isotope effects for the native enzyme from A. vinelandii remained constant as the pH was varied in the absence of MgATP. Secondary isotope effects with a poor substrate, dAMP, were 1.08 for both the mutant and wild type enzymes from A. vinelandii in the presence of allosteric activator. In the native enzyme, this isotope effect was independent of MgATP concentration. The relative insensitivity in the magnitude of observed isotope effects to Ph, allosteric activator, the mutant enzyme, and a poor substrate (dAMP) indicate that intrinsic isotope effects are being expressed. The data are interpreted in terms of a single rate-limiting transition state for hydrolysis of the N-glycosidic bond, althouth other mechanisms cannot be eliminated. Using this model, the transition states of the native A. vinelandii and E. coli enzymes exhibit properties of both dissociative and associative mechanisms but become more associative as the allosteric activator becomes saturating. The transition state of the mutant enzyme is more dissociative and is not altered by increasing concentrations of MgATP. Likewise, the transition state for dAMP hydrolysis does not change as a function of allosteric activation. A mechanism is proposed in which the allosteric activator causes the incipient nucleophile to be moved closer to the C-1' of AMP and induces additional strain on the N-glycosidic bond.

Original languageEnglish (US)
Pages (from-to)9418-9425
Number of pages8
JournalJournal of Biological Chemistry
Volume259
Issue number15
StatePublished - 1984
Externally publishedYes

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AMP nucleosidase
Isotopes
Chemical activation
Adenosine Triphosphate
Azotobacter vinelandii
Kinetics
Enzymes
Adenosine Monophosphate
Escherichia coli
Hydrolysis
Substrates
Enzyme Activators

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects of allosteric activation on the primary and secondary kinetic isotope effects for three AMP nucleosidases. / Parkin, D. W.; Schramm, Vern L.

In: Journal of Biological Chemistry, Vol. 259, No. 15, 1984, p. 9418-9425.

Research output: Contribution to journalArticle

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abstract = "Kinetic isotope effects (V/K) were measured with AMP nucleosidases isolated from Azotobacter vinelandii, from a V(max) mutant enzyme of A. vinelandii and from Escherichia coli. Specifically labeled AMP substrates were used to measure 3H secondary and 14C primary kinetic isotope effects on the N-glycosidic bond hydrolysis of AMP in the presence and absence of the allosteric activator, MgATP. Use of the three enzymes, variable MgATP concentration, a poor substrate (dAMP), and variable pH has allowed determination of the isotope effects over a 5000-fold range in the catalytic turnover number. The primary kinetic isotope effects were 1.025 ± 0.004 and 1.041 ± 0.006 for the native A. vindelandii enzyme and mutant enzyme, respectively, and were independent of MgATP concentration. The E. coli AMP nucleosidase had a primary isotope effect of 1.019 ± 0.003 which was also independent of MgATP concentration. The secondary kinetic isotope effect decreased from 1.066 ± 0.003 to 1.045 ± 0.002 for the native enzyme from A. vinelandii as the concentration of MgATP increased from 0 to 500 μM. The secondary isotope effect of the mutant ezyme remained constant at 1.088 ± 0.005 as the MgATP concentration increased from 0 to 500 μM. The secondary isotope effect of the E. coli enzyme showed a similar pattern to that of the native enzyme, decreasing from 1.087 ± 0.003 to 1.050 ± 0.003 as the enzyme was saturated with MgATP at a constant concentration of AMP. Saturation with AMP in the absence of MgATP gave similar results and suggested that AMP can cause the allosteric transition. Both the primary and secondary isotope effects for the native enzyme from A. vinelandii remained constant as the pH was varied in the absence of MgATP. Secondary isotope effects with a poor substrate, dAMP, were 1.08 for both the mutant and wild type enzymes from A. vinelandii in the presence of allosteric activator. In the native enzyme, this isotope effect was independent of MgATP concentration. The relative insensitivity in the magnitude of observed isotope effects to Ph, allosteric activator, the mutant enzyme, and a poor substrate (dAMP) indicate that intrinsic isotope effects are being expressed. The data are interpreted in terms of a single rate-limiting transition state for hydrolysis of the N-glycosidic bond, althouth other mechanisms cannot be eliminated. Using this model, the transition states of the native A. vinelandii and E. coli enzymes exhibit properties of both dissociative and associative mechanisms but become more associative as the allosteric activator becomes saturating. The transition state of the mutant enzyme is more dissociative and is not altered by increasing concentrations of MgATP. Likewise, the transition state for dAMP hydrolysis does not change as a function of allosteric activation. A mechanism is proposed in which the allosteric activator causes the incipient nucleophile to be moved closer to the C-1' of AMP and induces additional strain on the N-glycosidic bond.",
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AU - Schramm, Vern L.

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N2 - Kinetic isotope effects (V/K) were measured with AMP nucleosidases isolated from Azotobacter vinelandii, from a V(max) mutant enzyme of A. vinelandii and from Escherichia coli. Specifically labeled AMP substrates were used to measure 3H secondary and 14C primary kinetic isotope effects on the N-glycosidic bond hydrolysis of AMP in the presence and absence of the allosteric activator, MgATP. Use of the three enzymes, variable MgATP concentration, a poor substrate (dAMP), and variable pH has allowed determination of the isotope effects over a 5000-fold range in the catalytic turnover number. The primary kinetic isotope effects were 1.025 ± 0.004 and 1.041 ± 0.006 for the native A. vindelandii enzyme and mutant enzyme, respectively, and were independent of MgATP concentration. The E. coli AMP nucleosidase had a primary isotope effect of 1.019 ± 0.003 which was also independent of MgATP concentration. The secondary kinetic isotope effect decreased from 1.066 ± 0.003 to 1.045 ± 0.002 for the native enzyme from A. vinelandii as the concentration of MgATP increased from 0 to 500 μM. The secondary isotope effect of the mutant ezyme remained constant at 1.088 ± 0.005 as the MgATP concentration increased from 0 to 500 μM. The secondary isotope effect of the E. coli enzyme showed a similar pattern to that of the native enzyme, decreasing from 1.087 ± 0.003 to 1.050 ± 0.003 as the enzyme was saturated with MgATP at a constant concentration of AMP. Saturation with AMP in the absence of MgATP gave similar results and suggested that AMP can cause the allosteric transition. Both the primary and secondary isotope effects for the native enzyme from A. vinelandii remained constant as the pH was varied in the absence of MgATP. Secondary isotope effects with a poor substrate, dAMP, were 1.08 for both the mutant and wild type enzymes from A. vinelandii in the presence of allosteric activator. In the native enzyme, this isotope effect was independent of MgATP concentration. The relative insensitivity in the magnitude of observed isotope effects to Ph, allosteric activator, the mutant enzyme, and a poor substrate (dAMP) indicate that intrinsic isotope effects are being expressed. The data are interpreted in terms of a single rate-limiting transition state for hydrolysis of the N-glycosidic bond, althouth other mechanisms cannot be eliminated. Using this model, the transition states of the native A. vinelandii and E. coli enzymes exhibit properties of both dissociative and associative mechanisms but become more associative as the allosteric activator becomes saturating. The transition state of the mutant enzyme is more dissociative and is not altered by increasing concentrations of MgATP. Likewise, the transition state for dAMP hydrolysis does not change as a function of allosteric activation. A mechanism is proposed in which the allosteric activator causes the incipient nucleophile to be moved closer to the C-1' of AMP and induces additional strain on the N-glycosidic bond.

AB - Kinetic isotope effects (V/K) were measured with AMP nucleosidases isolated from Azotobacter vinelandii, from a V(max) mutant enzyme of A. vinelandii and from Escherichia coli. Specifically labeled AMP substrates were used to measure 3H secondary and 14C primary kinetic isotope effects on the N-glycosidic bond hydrolysis of AMP in the presence and absence of the allosteric activator, MgATP. Use of the three enzymes, variable MgATP concentration, a poor substrate (dAMP), and variable pH has allowed determination of the isotope effects over a 5000-fold range in the catalytic turnover number. The primary kinetic isotope effects were 1.025 ± 0.004 and 1.041 ± 0.006 for the native A. vindelandii enzyme and mutant enzyme, respectively, and were independent of MgATP concentration. The E. coli AMP nucleosidase had a primary isotope effect of 1.019 ± 0.003 which was also independent of MgATP concentration. The secondary kinetic isotope effect decreased from 1.066 ± 0.003 to 1.045 ± 0.002 for the native enzyme from A. vinelandii as the concentration of MgATP increased from 0 to 500 μM. The secondary isotope effect of the mutant ezyme remained constant at 1.088 ± 0.005 as the MgATP concentration increased from 0 to 500 μM. The secondary isotope effect of the E. coli enzyme showed a similar pattern to that of the native enzyme, decreasing from 1.087 ± 0.003 to 1.050 ± 0.003 as the enzyme was saturated with MgATP at a constant concentration of AMP. Saturation with AMP in the absence of MgATP gave similar results and suggested that AMP can cause the allosteric transition. Both the primary and secondary isotope effects for the native enzyme from A. vinelandii remained constant as the pH was varied in the absence of MgATP. Secondary isotope effects with a poor substrate, dAMP, were 1.08 for both the mutant and wild type enzymes from A. vinelandii in the presence of allosteric activator. In the native enzyme, this isotope effect was independent of MgATP concentration. The relative insensitivity in the magnitude of observed isotope effects to Ph, allosteric activator, the mutant enzyme, and a poor substrate (dAMP) indicate that intrinsic isotope effects are being expressed. The data are interpreted in terms of a single rate-limiting transition state for hydrolysis of the N-glycosidic bond, althouth other mechanisms cannot be eliminated. Using this model, the transition states of the native A. vinelandii and E. coli enzymes exhibit properties of both dissociative and associative mechanisms but become more associative as the allosteric activator becomes saturating. The transition state of the mutant enzyme is more dissociative and is not altered by increasing concentrations of MgATP. Likewise, the transition state for dAMP hydrolysis does not change as a function of allosteric activation. A mechanism is proposed in which the allosteric activator causes the incipient nucleophile to be moved closer to the C-1' of AMP and induces additional strain on the N-glycosidic bond.

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