Transition-state analysis of a V(max) mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

D. W. Parkin, F. Mentch, G. A. Banks, B. A. Horenstein, Vern L. Schramm

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Abstract

The transition state of the V(max) mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., and Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the K(m) for substrate, the activation constant for MgATP, and the K(i) for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 ± 0.002 to 1.021 ± 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 ± 0.003 to 1.094 ± 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 ± 0.003 to 1.070 ± 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 ± 0.006 and 1.089 ± 0.002 and were not changed by addition of MgATP. Transition-state analysis using bond-energy and bond-order vibrational analysis indicated that the transition state for the mutant enzyme has a similar position in the reaction coordinate compared to that for the normal enzyme. The mutant enzyme is less effective in stabilizing the carbocation-like intermediate and in the ability to protonate N7 of adenine to create a better leaving group. This altered transition-state structure was confirmed by an altered substrate specificity for the mutant protein.

Original languageEnglish (US)
Pages (from-to)4586-4594
Number of pages9
JournalBiochemistry
Volume30
Issue number18
StatePublished - 1991

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AMP nucleosidase
Adenosine Monophosphate
Isotopes
Atoms
Kinetics
Adenosine Triphosphate
Enzymes
Substrates
Chemical activation
Activator Appliances
Azotobacter vinelandii

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transition-state analysis of a V(max) mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects. / Parkin, D. W.; Mentch, F.; Banks, G. A.; Horenstein, B. A.; Schramm, Vern L.

In: Biochemistry, Vol. 30, No. 18, 1991, p. 4586-4594.

Research output: Contribution to journalArticle

Parkin, D. W. ; Mentch, F. ; Banks, G. A. ; Horenstein, B. A. ; Schramm, Vern L. / Transition-state analysis of a V(max) mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects. In: Biochemistry. 1991 ; Vol. 30, No. 18. pp. 4586-4594.
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abstract = "The transition state of the V(max) mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., and Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2{\%} of the rate of the normal enzyme with only minor changes in the K(m) for substrate, the activation constant for MgATP, and the K(i) for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 ± 0.002 to 1.021 ± 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 ± 0.003 to 1.094 ± 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 ± 0.003 to 1.070 ± 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 ± 0.006 and 1.089 ± 0.002 and were not changed by addition of MgATP. Transition-state analysis using bond-energy and bond-order vibrational analysis indicated that the transition state for the mutant enzyme has a similar position in the reaction coordinate compared to that for the normal enzyme. The mutant enzyme is less effective in stabilizing the carbocation-like intermediate and in the ability to protonate N7 of adenine to create a better leaving group. This altered transition-state structure was confirmed by an altered substrate specificity for the mutant protein.",
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AU - Banks, G. A.

AU - Horenstein, B. A.

AU - Schramm, Vern L.

PY - 1991

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N2 - The transition state of the V(max) mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., and Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the K(m) for substrate, the activation constant for MgATP, and the K(i) for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 ± 0.002 to 1.021 ± 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 ± 0.003 to 1.094 ± 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 ± 0.003 to 1.070 ± 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 ± 0.006 and 1.089 ± 0.002 and were not changed by addition of MgATP. Transition-state analysis using bond-energy and bond-order vibrational analysis indicated that the transition state for the mutant enzyme has a similar position in the reaction coordinate compared to that for the normal enzyme. The mutant enzyme is less effective in stabilizing the carbocation-like intermediate and in the ability to protonate N7 of adenine to create a better leaving group. This altered transition-state structure was confirmed by an altered substrate specificity for the mutant protein.

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