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An investigation of the electrophilic active site of histidine and phenylalanine ammonia-lyase

University of British Columbia

Histidine ammonia-lyase from Pseudomonas putida (hutH, EC 4.3.1.3) contains a unique active site electrophile that forms autocatalytically via a dehydrating backbone cyclization of the three amino acids alanine 142, serine 143 and glycine 144 and a second dehydration of the serine 143 side chain. The electrophile is a 4-methylidene-imidazole-5-one (MIO) that, following enzyme inactivation in the presence of cysteine and oxygen at pH 10.5, has been isolated as a component of a chromophoric octapeptide. The modified MIO is found to be an oxidized adduct of cysteine to which another cysteine or P-mercaptoethanol is linked through a disulfide bridge. One and twodimensional NMR experiments as well as high-resolution mass and UV absorbance spectra indicate that each MIO-containing octapeptide consists of at least three interconverting isomers, with a covalent linkage between the exocyclic MIO methylidene carbon and the amine of cysteine. This information and data from inactivation studies employing analogues of cysteine lacking an amino functionality suggest an inactivation mechanism that strongly supports the Retey mechanism of HAL with histidine. It is likely that the closely related enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.3) shares this mechanism. A fluorine-containing analogue of L-phenylalanine, erythro 3-fluorophenylalanine, was synthesized and was found to act as a competitive inhibition of PAL from poplar, with a Ki value of 45±5 µM. This is significantly smaller than the Km value of 250 µM for L-phenylalanine, indicating that erythro 3-fluorophenylalanine is a reasonably good competitive inhibitor of PAL. A variety of analogues of L-histidine were synthesized, including (S)-2-halo-3-(imidazol-4- yl) propionic acid, where halo is fluoro, chloro or bromo, and β-(pyrazol-l-yl)-L-alanine. These compounds were found to be neither substrates nor inhibitors of HAL. Additional experiments were performed on 4-methylimidazole/glycine and imidazole/L-alanine to test for isotope exchange in the imidazole ring but none were observed. 2-methylimidazole was found to be a competitive inhibitor of HAL with a Ki value of 20 mM.

Thesis/Dissertation

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2009-09-22

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http://hdl.handle.net/2429/12931

Chemistry

University of British Columbia

UBCV

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