C. Appert et al., STRUCTURAL AND CATALYTIC PROPERTIES OF THE 4 PHENYLALANINE AMMONIA-LYASE ISOENZYMES FROM PARSLEY (PETROSELINUM-CRISPUM NYM), European journal of biochemistry, 225(1), 1994, pp. 491-499
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 glutathion
e S transferase were expressed in Escherichia coli, purified and cleav
ed. All of the resulting phenylalanine ammonia-lyase proteins, as well
as the fusion proteins, were catalytically active. The turnover numbe
r 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 differentia
l expression in various parts of parsley plants, the four phenylalanin
e ammonia-lyase isoenzymes exhibited very similar apparent K-m values
for L-phenylalanine (15-24.5 mu M) as well as identical temperature (5
8 degrees C) and pH (8.5) optima. All of them were competitively inhib
ited by (E)-cinnamate with similar efficiency (K-i values: 9.1-21.5 mu
M), lacked cooperative behaviour, and accepted L-tyrosine as a substr
ate 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.