IDENTIFICATION OF ESSENTIAL AMINO-ACIDS IN PHENYLALANINE AMMONIA-LYASE BY SITE-DIRECTED MUTAGENESIS

The postulated precursor of the prosthetic dehydroalanine of phenylala nine ammonia-lyase (PAL), serine 202, was changed to cysteine by site- directed mutagenesis. After cloning and heterologous expression in Esc herichia coli, the gene product was assayed for PAL activity. Mutant S 202C showed full catalytic activity, and its kinetic constants and the amount of thiol groups were identical to those of wild-type PAL. It m ust be concluded that in a posttranslational modification both water a nd hydrogen sulfide can be eliminated from the amino acid in position 202 to form dehydroalanine. In an attempt to identify further amino ac ids essential either for the posttranslational modification or for cat alysis, arginine 174, glutamine 425, and lysine 499 were changed to is oleucine. Analysis of the heterologously expressed mutated gene produc ts revealed that only the R174I mutant showed a significantly lower V- max value (1/450) identifying this arginine as important. This finding was supported by treatment of wild-type PAL and mutant R174I with phe nylglyoxal and 2,3-butandione. Both react specifically with the guanid ino group of arginine. They irreversibly inhibited wild-type PAL but h ad no influence of the V-max value of mutant R174I. Preincubation with L-phenylalanine protected wild-type PAL from inhibition by phenylglyo xal indicating that arginine 174 is close to the active site. Incubati on with KCN irreversibly abolished the remaining activity of mutant R1 74I leading to the conclusion that arginine 174 is important in cataly sis.