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Kinetic Analysis of the Reactions Catalyzed by Histidine and Phenylalanine Ammonia Lyases
Author(s) -
Viergutz Sandra,
Rétey János
Publication year - 2004
Publication title -
chemistry and biodiversity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 70
eISSN - 1612-1880
pISSN - 1612-1872
DOI - 10.1002/cbdv.200490026
Subject(s) - chemistry , phenylalanine , kinetic isotope effect , histidine , electrophile , ammonia , stereochemistry , catalysis , enzyme kinetics , substrate (aquarium) , deuterium , phenylalanine ammonia lyase , enzyme , medicinal chemistry , active site , organic chemistry , biochemistry , amino acid , physics , oceanography , quantum mechanics , geology
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 (2 S ,3 S )‐[3‐ 2 H 1 ]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 k H / k D 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 ( k H / k D =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‐4 H ‐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 enzymeammonia complex was formed, and the mutant behaved upon addition of ( E )‐[ 14 C]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 ).