Open AccessAn active site homology model of phenylalanine ammonia‐lyase from P. crispum
Author(s) -
Röther Dagmar,
Poppe László,
Morlock Gaby,
Viergutz Sandra,
Rétey János
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.02984.x
Subject(s) - active site , phenylalanine ammonia lyase , enzyme kinetics , homology modeling , site directed mutagenesis , histidine , amino acid , lyase , enzyme , phenylalanine , mutant , stereochemistry , mutagenesis , biochemistry , chemistry , homology (biology) , biology , gene
The plant enzyme phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) 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. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in k cat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345‐, 615‐ and 14‐fold lower k cat , respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild‐type PAL. To elucidate the possible function of the mutated amino‐acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.