Transition-state analysis of AMP deaminase.
The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been characterized by 14C and 15N Vmax/Km heavy-atom kinetic isotope effects. The primary 6-14C isotope effect was measured with [6-14C]AMP, and the 6-15N primary isotope effect was measured by isotope ratio mass spectrometry using the natural abundance of 15N in AMP and by using 15N release from ATP as a slow substrate. Isotope effects for AMP as the substrate were measured in the presence and absence of ATP as an allosteric activator and GTP as an allosteric inhibitor. Kinetic isotope effects with [6-14C]AMP were 1.030 +/- 0.003, 1.038 +/- 0.004, and 1.042 +/- 0.003 in the absence of effectors and in the presence of ATP and GTP, respectively. Isotope effects for [6-15N]AMP averaged 1.010 +/- 0.002. Allosteric activation increased the 15N isotope effect to 1.016 +/- 0.003. A primary 15N kinetic isotope effect with ATP, which has a Vmax/Km 10(-6) that for AMP, was 1.013 +/- 0.001. The presence of D2O as solvent caused a marginally significant decrease in the [6-15N]AMP kinetic isotope effect from 1.011 +/- 0.001 to 1.007 +/- 0.002. Previous studies have established that the solvent D2O effect is inverse (0.34) for slow substrates with two or more protons transferred prior to transition state formation and remains inverse (0.79) with AMP as substrate [Merkler, D. J., & Schramm, V. L. (1993) Biochemistry 32, 5792-5799]. Bond vibrational analysis was used to identify transition states for AMP deaminase that are consistent with all kinetic isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Merkler
D J
DJ
Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461.
Kline
P C
PC
Weiss
P
P
Schramm
V L
VL
D000659:AMP Deaminase / Q000737:chemistry*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D005656:Fungal Proteins / Q000737:chemistry; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D005656:Fungal Proteins; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000659:AMP Deaminase