Catalytic mechanism of yeast adenosine 5′-monophosphate deaminase. Zinc content, substrate specificity, pH studies, and solvent isotope effects

David J. Merkler, Vern L. Schramm

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Abstract

Adenosine 5′-monophosphate (AMP) deaminase from baker's yeast is an allosteric enzyme containing a single AMP binding site and two ATP regulatory sites per polypeptide [Merkler, D. J., & Schramm, V. L. (1990) j. Biol Chem. 265, 4420-4426]. The enzyme contains 0.98 ± 0.17 zinc atom per subunit. The X-ray crystal structure for mouse adenosine deaminase shows zinc in contact with the attacking water nucleophile using purine riboside as a transition-state inhibitor [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284]. Alignment of the amino acid sequence for yeast AMP deaminase with that for mouse adenosine deaminase demonstrates conservation of the amino acids known from the X-ray crystal structure to bind to the zinc and to a transition-state analogue. On the basis of these similarities, yeast AMP deaminase is also proposed to use a Zn2+-activated water molecule to attack C6 of AMP with the displacement of NH3. The pKm and pKi profiles for AMP and a competitive inhibitor overlap in a bell-shaped curve with pKa values of 7.0 and 7.4. This pattern is characteristic of a rapid equilibrium between AMP and the enzyme, thus confirming the rapid equilibrium random kinetic patterns [Merkler, D. J., Wali, A. S., Taylor, J., Schramm, V. L. (1989) J. Biol. Chem. 264, 21422-21430]. The Vmax of the reaction requires one unprotonated and one protonated group with pKa values of 6.4 ± 0.2 and 7.7 ± 0.3, respectively. The 2H2O-induced shifts of the pKa values for these groups are consistent with a carboxylate and a histidine, groups known to be in contact with purine riboside in the adenosine deaminase structure. The Vmax/Km profile is similar except that the pKa values are 6.7 and 7.3, respectively. Kinetic studies with 2H2O as solvent gave inverse Vmax/Km solvent deuterium isotope effects, i.e., reaction rates more rapid in 2H2O than H2O. Solvent 2H2O isotope effects varied from 0.79 ± 0.11 to 0.33 ± 0.03, with the slowest substrates giving the largest isotope effects. Proton inventory studies with a slow substrate indicated that two or more protons give rise to the solvent isotope effect. The results are interpreted in a mechanism where equilibrium proton transfers from the zinc-bound water and/or a compressed hydrogen bond to the substrate contribute to the observed inverse solvent isotope effect.

Original languageEnglish (US)
Pages (from-to)5792-5799
Number of pages8
JournalBiochemistry
Volume32
Issue number22
StatePublished - 1993

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Adenosine Monophosphate
Substrate Specificity
Isotopes
Yeast
Adenosine
Zinc
Yeasts
Substrates
Protons
Water
Enzymes
Crystal structure
X-Rays
Amino Acids
X rays
Nucleophiles
Kinetics
Adenosine Deaminase
Proton transfer
Deuterium

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{b70ca4dc15e84139ba8b8ba5b4f0cb4b,
title = "Catalytic mechanism of yeast adenosine 5′-monophosphate deaminase. Zinc content, substrate specificity, pH studies, and solvent isotope effects",
abstract = "Adenosine 5′-monophosphate (AMP) deaminase from baker's yeast is an allosteric enzyme containing a single AMP binding site and two ATP regulatory sites per polypeptide [Merkler, D. J., & Schramm, V. L. (1990) j. Biol Chem. 265, 4420-4426]. The enzyme contains 0.98 ± 0.17 zinc atom per subunit. The X-ray crystal structure for mouse adenosine deaminase shows zinc in contact with the attacking water nucleophile using purine riboside as a transition-state inhibitor [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284]. Alignment of the amino acid sequence for yeast AMP deaminase with that for mouse adenosine deaminase demonstrates conservation of the amino acids known from the X-ray crystal structure to bind to the zinc and to a transition-state analogue. On the basis of these similarities, yeast AMP deaminase is also proposed to use a Zn2+-activated water molecule to attack C6 of AMP with the displacement of NH3. The pKm and pKi profiles for AMP and a competitive inhibitor overlap in a bell-shaped curve with pKa values of 7.0 and 7.4. This pattern is characteristic of a rapid equilibrium between AMP and the enzyme, thus confirming the rapid equilibrium random kinetic patterns [Merkler, D. J., Wali, A. S., Taylor, J., Schramm, V. L. (1989) J. Biol. Chem. 264, 21422-21430]. The Vmax of the reaction requires one unprotonated and one protonated group with pKa values of 6.4 ± 0.2 and 7.7 ± 0.3, respectively. The 2H2O-induced shifts of the pKa values for these groups are consistent with a carboxylate and a histidine, groups known to be in contact with purine riboside in the adenosine deaminase structure. The Vmax/Km profile is similar except that the pKa values are 6.7 and 7.3, respectively. Kinetic studies with 2H2O as solvent gave inverse Vmax/Km solvent deuterium isotope effects, i.e., reaction rates more rapid in 2H2O than H2O. Solvent 2H2O isotope effects varied from 0.79 ± 0.11 to 0.33 ± 0.03, with the slowest substrates giving the largest isotope effects. Proton inventory studies with a slow substrate indicated that two or more protons give rise to the solvent isotope effect. The results are interpreted in a mechanism where equilibrium proton transfers from the zinc-bound water and/or a compressed hydrogen bond to the substrate contribute to the observed inverse solvent isotope effect.",
author = "Merkler, {David J.} and Schramm, {Vern L.}",
year = "1993",
language = "English (US)",
volume = "32",
pages = "5792--5799",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "22",

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TY - JOUR

T1 - Catalytic mechanism of yeast adenosine 5′-monophosphate deaminase. Zinc content, substrate specificity, pH studies, and solvent isotope effects

AU - Merkler, David J.

AU - Schramm, Vern L.

PY - 1993

Y1 - 1993

N2 - Adenosine 5′-monophosphate (AMP) deaminase from baker's yeast is an allosteric enzyme containing a single AMP binding site and two ATP regulatory sites per polypeptide [Merkler, D. J., & Schramm, V. L. (1990) j. Biol Chem. 265, 4420-4426]. The enzyme contains 0.98 ± 0.17 zinc atom per subunit. The X-ray crystal structure for mouse adenosine deaminase shows zinc in contact with the attacking water nucleophile using purine riboside as a transition-state inhibitor [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284]. Alignment of the amino acid sequence for yeast AMP deaminase with that for mouse adenosine deaminase demonstrates conservation of the amino acids known from the X-ray crystal structure to bind to the zinc and to a transition-state analogue. On the basis of these similarities, yeast AMP deaminase is also proposed to use a Zn2+-activated water molecule to attack C6 of AMP with the displacement of NH3. The pKm and pKi profiles for AMP and a competitive inhibitor overlap in a bell-shaped curve with pKa values of 7.0 and 7.4. This pattern is characteristic of a rapid equilibrium between AMP and the enzyme, thus confirming the rapid equilibrium random kinetic patterns [Merkler, D. J., Wali, A. S., Taylor, J., Schramm, V. L. (1989) J. Biol. Chem. 264, 21422-21430]. The Vmax of the reaction requires one unprotonated and one protonated group with pKa values of 6.4 ± 0.2 and 7.7 ± 0.3, respectively. The 2H2O-induced shifts of the pKa values for these groups are consistent with a carboxylate and a histidine, groups known to be in contact with purine riboside in the adenosine deaminase structure. The Vmax/Km profile is similar except that the pKa values are 6.7 and 7.3, respectively. Kinetic studies with 2H2O as solvent gave inverse Vmax/Km solvent deuterium isotope effects, i.e., reaction rates more rapid in 2H2O than H2O. Solvent 2H2O isotope effects varied from 0.79 ± 0.11 to 0.33 ± 0.03, with the slowest substrates giving the largest isotope effects. Proton inventory studies with a slow substrate indicated that two or more protons give rise to the solvent isotope effect. The results are interpreted in a mechanism where equilibrium proton transfers from the zinc-bound water and/or a compressed hydrogen bond to the substrate contribute to the observed inverse solvent isotope effect.

AB - Adenosine 5′-monophosphate (AMP) deaminase from baker's yeast is an allosteric enzyme containing a single AMP binding site and two ATP regulatory sites per polypeptide [Merkler, D. J., & Schramm, V. L. (1990) j. Biol Chem. 265, 4420-4426]. The enzyme contains 0.98 ± 0.17 zinc atom per subunit. The X-ray crystal structure for mouse adenosine deaminase shows zinc in contact with the attacking water nucleophile using purine riboside as a transition-state inhibitor [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284]. Alignment of the amino acid sequence for yeast AMP deaminase with that for mouse adenosine deaminase demonstrates conservation of the amino acids known from the X-ray crystal structure to bind to the zinc and to a transition-state analogue. On the basis of these similarities, yeast AMP deaminase is also proposed to use a Zn2+-activated water molecule to attack C6 of AMP with the displacement of NH3. The pKm and pKi profiles for AMP and a competitive inhibitor overlap in a bell-shaped curve with pKa values of 7.0 and 7.4. This pattern is characteristic of a rapid equilibrium between AMP and the enzyme, thus confirming the rapid equilibrium random kinetic patterns [Merkler, D. J., Wali, A. S., Taylor, J., Schramm, V. L. (1989) J. Biol. Chem. 264, 21422-21430]. The Vmax of the reaction requires one unprotonated and one protonated group with pKa values of 6.4 ± 0.2 and 7.7 ± 0.3, respectively. The 2H2O-induced shifts of the pKa values for these groups are consistent with a carboxylate and a histidine, groups known to be in contact with purine riboside in the adenosine deaminase structure. The Vmax/Km profile is similar except that the pKa values are 6.7 and 7.3, respectively. Kinetic studies with 2H2O as solvent gave inverse Vmax/Km solvent deuterium isotope effects, i.e., reaction rates more rapid in 2H2O than H2O. Solvent 2H2O isotope effects varied from 0.79 ± 0.11 to 0.33 ± 0.03, with the slowest substrates giving the largest isotope effects. Proton inventory studies with a slow substrate indicated that two or more protons give rise to the solvent isotope effect. The results are interpreted in a mechanism where equilibrium proton transfers from the zinc-bound water and/or a compressed hydrogen bond to the substrate contribute to the observed inverse solvent isotope effect.

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