Tritium secondary kinetic isotope effect on phenylalanine ammonia-lyase-catalyzed reaction

The mechanism by which phenylalanine ammonialyase (PAL, EC 4.3.1.5) catalyz es the reversible elimination of ammonia from phenylalanine yielding (E)-ci nnamic acid has gained much attention in the recent years. Dehydroalanine i s essential for the catalysis. It was assumed that this prostetic group act s as the electrophile, leading to a covalently bonded enzyme-intermediate c omplex with quarternary nitrogen of phenylalanine. Recently, an alternative mechanism has been suggested in which the enzyme-intermediate complex is f ormed in a Friedel-Crafts reaction between dehydroalanine and orthocarbon o f the aromatic ring. Using semiempirical calculations we have shown that th ese two alternative mechanisms can be distinguished on the basis of the hyd rogen secondary kinetic isotope effect when tritium label is placed in the orthopositions. Our calculations indicated also that the kinetic isotope ef fect measured using ring-labeled d(5)-phenylalanine could not be used to di fferentiate these alternative mechanisms. Measured secondary tritium kineti c isotope effect shows strong dependence on the reaction progress, starting at the inverse value of k(H)/k(T) = 0.85 for 5% conversion and reaching th e normal value of about 1.15 as the conversion increases to 20%. This depen dence has been interpreted in terms of a complex mechanism with initial for mation of the Friedel-Crafts type intermediate. (C) 1999 Academic Press.