tailieunhanh - Báo cáo Y học: An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum

The plant enzyme phenylalanine ammonia-lyase (PAL, EC ) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138 | Eur. J. Biochem. 269 3065-3075 2002 FEBS 2002 doi An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum Dagmar Rother1 László Poppe2 Gaby Morlock1 Sandra Viergutz1 and Janos Retey1 Institute for Organic Chemistry University of Karlsruhe Germany 2Institute for Organic Chemistry Budapest University of Technology and Economics Hungary The plant enzyme phenylalanine ammonia-lyase PAL EC shows homology to histidine ammonia-lyase HAL whose structure has been solved by X-ryy crytta--lography. Based on amino-acid ssquence iilignmenl of the two enzymes mutagenesis was performed on mmno-ccid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the noOovnng nrmno-ccic resíduSs S203 14354. Y110 Y351 N260 Q348 F400 Q488 and L138. Dtter-mination of the kinetic constants of the overexpressed and purified en ymes It ve ae d than mutahonesis led in chh sese to diminished activity. Mutants S203A R354A and Y351F showed a deeteesc in k by factors of 435 130 and . respectively. Mutants F400A Q433A and L138H showed a 345- 615- and 14-fodd lower k respectively. The greatest loss of activity occurredin the PAL mutants N260A Q343A and Y110F whích were 7000. 23300 nnd 70 000 times Isss active than wild-type PAL. To elucidate the possible function of the mutatedamino-acidresidues in PAL we built a homology model of PAL based on structural data of HAL andmutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowedus to propose possible roles for the corresponding residues in PAL catalysis. Keywords phenylalanine ammonia-lyase PAL MIO site-directed mutagenesis homology model. Phenylalanine ammonia-lyase PAL EC is a very important plant enzyme that catalyses the conversion of L-phenylalanine into .