The behavior of substrate analogues and secondary deuterium isotope effects in the phenylalanine ammonia-lyase reaction

Metacresol and glycine can be thought as a dissection of metatyrosine, which is an excellent substrate of phenylalanine ammonia-lyase (PAL) (B. Schuster and J. Retey, PNAS 92, 8433, 1995). Whereas metacresol was a very weak inhibitor and glycine was inert, simultaneous addition of both compounds led to synergistic inhibition of PAL. [H-2(5)]Phenylalanine as a substrate showed a kinetic deuterium isotope effect of 9% (k(H)/k(2H) = 1.09 +/- 0.01) while its K-m value was identical to that of the unlabeled substrate. The following substrate analogues were synthesized and assayed with PAL: cyclooctatetraenyl (COT)-D,L)-alanine as well as 2-pyridyl-, 3-pyridyl-, and 4-pyridyl-(L)-alanines. While COT-(D,L)-alanine turned out to be a rather reluctant substrate, all three isomers of pyridyl-(L)-alanines were converted with a comparable or even higher V-max than L-phenylalanine into the corresponding pyridyl acrylic acids. Their K-m values were, however, an order of magnitude higher than that of the natural substrate. These results are discussed in terms of the novel mechanism which implies an electrophilic attack of the prosthetic dehydroalanine at the aromatic ring. The heats of formation of the putative sigma complexes of the electrophilic substitution at the pyridine ring have been calculated using semiempirical force-field methods. The results show the feasibility of the proposed mechanism also with the substrate analogues. (C) 1998 Academic Press.