Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase

The authors have determined 15N isotope effects and solvent deuterium isotope effects for adenosine deaminase using both adenosine and the slow alternate substrate 7,8-dihydro-8-oxoadenosine. With adenosine, 15N isotope effects were 1.0040 in H2O and 1.0023 in D2O, and the solvent deuterium isotope effect was 0.77. With 7,8-dihydro-8-oxoadenosine, 15N isotope effects were 1.015 in H2O and 1.0131 in D2O, and the solvent deuterium isotope effect was 0.45. The inverse solvent deuterium isotope effect shows that the fractionation factor of a proton, which is originally < 0.6, increases to near unity during formation of the tetrahedral intermediate from which ammonia is released. Proton inventories for 1/V and 1/(V/K) vs percent D2O are linear, indicating that a single proton has its fractionation factor altered during the reaction. They conclude that a sulfhydryl group on the enzyme donates its proton to oxygen or nitrogen during this step. pH profiles with 7,8-dihydro-8-oxoadenosine suggest that the pK of this sulfhydryl group is 8.45. The inhibition of adenosine deaminase by cadmium also shows a pK of ∼ 9 from the pK/sub i/ profile. Quantitative analysis of the isotope effects suggest an intrinsic 15N isotope effect for the release of ammonia from the tetrahedral intermediate. The most likely chemical mechanism for adenosine deaminsase involves protonation of N-1 by the sulfhydryl group while water adds to C-6 with general base assistance by a histidine with pK = 5.24. The pK values for 7,8-dihydro-8-oxoadenosine are 3.0 and 8.8 and for 7,8-dihydro-8-oxoinosine 7.8 and 11.1. The shape of a plot of (V/K)K/sub i/ vs pH is shown to be a very sensitive criterion for the stickiness of a substrate, and by this test adenosine is not sticky