STRUCTURAL AND CATALYTIC PROPERTIES OF THE 4 PHENYLALANINE AMMONIA-LYASE ISOENZYMES FROM PARSLEY (PETROSELINUM-CRISPUM NYM)

Near-full-length cDNAs for the four phenylalanine ammonia-lyase (PAL) isoenzymes in parsley (Petroselinum crispum Nym.) were cloned and the complete amino acid sequences deduced. Fusion proteins with glutathione S transferase were expressed in Escherichia coli, purified and cleaved. All of the resulting phenylalanine ammonia-lyase proteins, as well as the fusion proteins, were catalytically active. The turnover number of one selected isoenzyme, PAL-1, was estimated to be around 22 s(-1) for each active site. In contrast to a certain degree of differential expression in various parts of parsley plants, the four phenylalanine ammonia-lyase isoenzymes exhibited very similar apparent K-m values for L-phenylalanine (15-24.5 mu M) as well as identical temperature (58 degrees C) and pH (8.5) optima. All of them were competitively inhibited by (E)-cinnamate with similar efficiency (K-i values: 9.1-21.5 mu M), lacked cooperative behaviour, and accepted L-tyrosine as a substrate with low affinity (K-m values: 2.6-7.8 mM). These results suggest that the occurrence of multiple gene copies has a function other than encoding isoenzymes with different enzyme kinetic properties.