Use of nitrogen-15 and deuterium isotope effects to determine the chemical mechanism of phenylalanine ammonia-lyase

Publisher Website
Google Scholar
Abstract
Phenylalanine ammonia-lyase was shown to catalyze the elimination of NH3 from the slow alternate substrate 3-(1,4-cyclohexadienyl)alanine by E1 cb mechanism with a carbanion intermediate. This conclusion resulted from comparison of 15N isotope effects with deuterated (0.9921) and unlabeled substrates (1.0047), and a deuterium (D) isotope effect of 2.0 from dideuteration at C-3, with the equations for concerted, carbanion, and carbonium ion mechanisms. The 15N equilibrium isotope effect on the addition of the substrate to the dehydroalanine prosthetic group of the enzyme is 0.979, while the kinetic 15N isotope effect on the reverse of this step is 1.03-1.04 and the intrinsic D isotope effect on proton removal is in the range 4-6. Isotope effects with phenylalanine itself are small (15N ones of 1.0021 and 1.0010 when unlabeled or 3-dideuterated and a D isotope effect of 1.15) but are consistent with the same mechanism with drastically increased commitments, including a sizable external one (i.e., phenylalanine is sticky). pH profiles show that the amino group of the substrate must be unprotonated to react but that a group on the enzyme with a pK of 9 must be protonated, possibly to catalyze addition of the substrate to dehydroalanine. Incorrectly protonated enzyme-substrate complexes do not form. Equilibrium 15N isotope effects are 1.016 for the deprotonation of phenylalanine or its cyclohexadienyl analogue, 1.0192 for deprotonation of NH4+, 1.0163 for the conversion of the monoanion of phenylalanine to NH3, and 1.0138 for the conversion of the monoanion of aspartate to NH4+. The value of 1.0246 for the deprotonation of ND4+ in D2O shows that replacing an N.sbd.H with an N.sbd.D bond raises the 15N fractionation factor by 0.5%.