Transition-State Analysis of AMP Deaminase

The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been characterized by ¹⁴C and ¹⁵N V_max/K_m 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 V_max./K_m 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. Fully concerted reaction mechanisms can be eliminated, since these would result in normal D₂O solvent isotope effects and are inconsistent with ¹⁴C and ¹⁵N kinetic isotope effects. The transition state most consistent with the data is characterized by an attacking hydroxyl with a bond order near 0.8, a fully bonded NH₂, nearly complete conversion to sp³ at C6, and highly asymmetric, nearly complete protonation of N1. This transition state leads to the formation of a short-lived tetrahedral intermediate. Formation of the tetrahedral intermediate is slow, while protonation of NH₂ in the intermediate and departure of NH₃ occur in rapid steps.