Structural and catalytic properties of the four phenylalanine ammonia-lyase isoenzymes from parsley (Petroselinum crispum Nym.).
Near-full-length cDNAs for the four phenylalanine ammonia-lyase (PAL) isoenzymes in parsley (Petroselium 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 Km values for L-phenylalanine (15-24.5 microM) 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 (Ki values: 9.1-21.5 microM), lacked cooperative behaviour, and accepted L-tyrosine as a substrate with low affinity (Km 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.
Appert
C
C
Institut für Pflanzenwissenschaften, Eidgenössische Technische Hochschule Zürich, Switzerland.
Logemann
E
E
Hahlbrock
K
K
Schmid
J
J
Amrhein
N
N
D000595:Amino Acid Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D019684:Magnoliopsida / Q000201:enzymology*; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase