Author(s): Kordell Welch, Adam Jones, Taylor Strain, Andrew Martineau
Mentor(s): Elizabeth Pierce, Mackay Steffensen
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 the elimination of the alpha-amino group from histidine. The way that HAL breaks down histidine is currently thought to follow one of two possible reaction pathways. In one possible mechanism, the substrate’s alpha amine attacks the MIO cofactor’s electrophilic carbon, making the amine a better leaving group as the substrate’s beta carbon is deprotonated. In the alternative mechanism, attack of the aromatic ring on the enzyme’s electrophilic carbon decreases the pKa of the substrate’s beta proton, allowing for deprotonation and elimination of ammonia. Computational, structural, and inhibition studies provide support for each mechanism. HAL from Pseudomonas putida has been shown to be reversibly inactivated by L-cysteine, which an x-ray crystal structure shows as covalently attached to the electrophilic group in the active site via its nitrogen atom. Using L-histidine as the lead compound, we have designed and synthesized various inhibitors changing the imidazole ring or the alpha-amino group to assess activity with different functional groups. We are currently working on enzyme kinetics using these predicted inhibitors to probe important interactions between HAL and its substrates to better understand the mechanism by which HAL functions.