THE MECHANISM OF ACTION OF PHENYLALANINE AMMONIA-LYASE - THE ROLE OF PROSTHETIC DEHYDROALANINE

Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslati onally modified by dehydrating its Ser-202 to the catalytically essent ial dehydroalanine prosthetic group, The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis, These mutants and the recombinant wild-type enzyme, after treatment with sod ium borohydride, were virtually inactive with L-phenylalanine as subst rate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants react ed about 20 times slower with L-4-nitrophenylalanine than the wild-typ e enzyme, their V-max for L-4-nitrophenylalanine was two orders of mag nitude higher than for L-phenylalanine, In contrast to L-tyrosine, whi ch was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the cr ucial step is an electrophilic attack of the prosthetic group at posit ion 2 or 6 of the phenyl group. In the resulting carbenium ion, the be ta-H-Si atom is activated in a similar way as it is in the nitro analo gue, Subsequent elimination of ammonia, concomitant with restoration o f both the aromatic ring and the prosthetic group, completes the catal ytic cycle.