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.

Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) [Schwede, T.F., Rétey, J., Schulz, G.E. (1999) Biochemistry, 38, 5355-5361]. To evaluate the importance of several amino-acid residues at the active site for substrate binding and catalysis, we mutated the following amino-acid codons in the HAL gene: R283, Y53, Y280, E414, Q277, F329, N195 and H83. Kinetic measurements with the overexpressed mutants showed that all mutations resulted in a decrease of catalytic activity. The mutants R283I, R283K and N195A were approximately 1640, 20 and 1000 times less active, respectively, compared to the single mutant C273A, into which all mutations were introduced. Mutants Y280F, F329A and Q277A exhibited approximately 55, 100 and 125 times lower activity, respectively. The greatest loss of activity shown was in the HAL mutants Y53F, E414Q, H83L and E414A, the last being more than 20 900-fold less active than the single mutant C273A, while H83L was 18 000-fold less active than mutant C273A. We propose that the carboxylate group of E414 plays an important role as a base in catalysis. To investigate a possible participation of active site amino acids in the formation of MIO, we used the chromophore formation upon treatment of HAL with l-cysteine and dioxygen at pH 10.5 as an indicator. All mutants, except F329A showed the formation of a 338-nm chromophore arising from a modified MIO group. The UV difference spectra of HAL mutant F329A with the MIO-free mutant S143A provide evidence for the presence of a MIO group in HAL mutant F329A also. For modelling of the substrate arrangement within the active site and protonation state of MIO, theoretical calculations were performed.

N‐Methyl‐L‐phenylalanine (5), N‐methyl‐4‐nitro‐L‐phenylalanine (6), and N,N‐dimethyl‐4‐nitro‐L‐phenylalanine (7⋅H+) were investigated as substrates or inhibitors of phenylalanine ammonia lyase from Petroselinum crispum. Whereas the former was a reluctant substrate (Km =6.6 mM, kcat =0.22 s−1), no reverse reaction could be detected by using methylamine and (E)‐cinnamate (2). The Km value for ammonia in the reverse reaction by using (E)‐cinnamate (2) was determined to be 4.4 and 2.6M at pH 8.8 and 10, respectively. The N‐methylated 4‐nitro‐L‐phenylalanines 6 and 7 showed only strong inhibitory effects (Ki =130 nM and 8 nM, resp.). These and former results are discussed in terms of the mechanism of action of phenyalalanine and histidine ammonia lyases.

Although both the structures and the reactions of histidine and phenylalanine ammonia lyases (HAL and PAL) are very similar, the former shows a primary kinetic deuterium (D) isotope effect, while the latter does not. In the HAL reaction, the release of ammonia is partially rate-determining and is slower than the release of the product (E)-urocanate (4), whereas in the PAL reaction, the release of (E)-cinnamate (2) is the rate-limiting step. With (2S,3S)-[3-(2)H1]phenylalanine (1a), we determined the kinetic D isotope effects with the PAL mutants Q487A, Y350F, L137 H, and the double mutant L137 H/Q487E. The kH/kD values for the former two were of the same magnitude as with wild-type PAL (1.20+/-0.07), while the exchange of L137 to H almost doubled the effect (kH/kD=2.32+/-0.01). We conclude that L137 is part of the hydrophobic pocket harboring the phenyl group of the substrate/product and is responsible for its strong binding. The stability of the HAL ammonia complex was demonstrated 40 years ago. Here, we show that, in contrast to the former assumption, ammonia in the complex is not covalently bound to the prosthetic electrophile, 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO; 5). We carried out experiments with a mutant enzyme lacking MIO and exhibiting ca. 10(3) times less activity. Nevertheless, the enzyme-ammonia complex was formed, and the mutant behaved upon addition of (E)-[14C]urocanate (4a) like wild-type HAL. We conclude, therefore, that ammonia is bound in the complex by Coulomb forces as ammonium ion and can be released only after (E)-urocanate (4).