Author(s): Romina Peralta, Isabel Amaro, Jessica Munro
Mentor(s): Elizabeth Pierce
Institution SUU
Histidine ammonia-lyase (HAL) is an enzyme that participates in the degradation pathway of L-histidine by catalyzing the elimination of the alpha-amino group to form trans-urocanate. In its active site, HAL contains an unusual cofactor 4-methylidene-imidazol-5-one (MIO) that is formed by post-translational modification. This modification is a backbone cyclization involving three residues (Ala-Ser-Gly) that form a conjugated imidazolone ring. The dehydration of the second residue, serine, forms dehydroalanine, with the alpha carbon as part of the backbone ring. The beta carbon of the dehydroalanine residue acts as an electrophile in eliminating the alpha-amino group from histidine. The positioning of this active site of HAL is situated in a broad, solvent-exposed pocket; suggesting ample room near the MIO group to enable substrate binding. The lack of aromaticity in MIO and localization of the tertiary nitrogens lone pair are thought to contribute to the sheer electrophilicity of the prosthetic group. This is supported by enzyme inactivation upon treatment with a strong nucleophile, as has been demonstrated in studies by Rӧther et al. (2001) and Givot et al. (1969). Experimental and computational analysis has additionally proposed the presence of metal ions to be valuable in enzyme catalysis, in combination with thiol reductants, although different researchers have produced contradictory results about the importance of metal ions and the effects of metal chelators on the enzyme’s activity. We have endeavored to introduce different divalent metals to HAL assays to further clarify the role of the metal ion and redox state of the protein in the reaction mechanism. Our approach to studying this is via measuring kinetic properties of the enzyme in the presence of different metals and reductants.