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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42 Scopus citations

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
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry

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