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Studies on the Mechanism of Action and the Structure of the Electrophilic Center of Histidine Ammonia Lyase

From the ¹ From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154

A procedure for the purification of highly purified l-histidine ammonia lyase is described. The enzyme exists in both an oxidized and reduced form. These forms are interconvertible and separable by electrophoresis. Studies with inhibitors suggest that the enzyme interacts with the substrate specifically through the carboxyl and amino groups and the imidazole moiety. Effective inhibitors contain functional groups corresponding to these three binding sites, while compounds with only two of the functional groups are poor inhibitors. A notable exception is glycine, which is a strong inhibitor. It was postulated that the binding of the substrate, as well as those inhibitors which contain a carbon atom corresponding to the ß carbon atom of histidine, involves strain or distortion induced by the enzyme. Therefore in the case of glycine, where no distortion is possible, the two functional groups provide sufficient interaction energy for binding to the enzyme. It was also shown that H_(s). bound to the ß carbon atom of histidine, is eliminated in the conversion to urocanic acid, and it was concluded that histidine ammonia lyase catalyzes a trans-elimination.

Exposure of the enzyme to nitromethane leads to loss of catalytic activity and to the covalent binding of approximately 2 moles of nitromethane per mole of enzyme. The extent of inactivation by nitromethane can be reduced by the addition of substrate or inhibitors, suggesting that nitromethane reacts at the active site. After catalytic reduction and acid hydrolysis of the nitromethane-inactivated enzyme, 2,4-diaminobutyric acid, 4-amino-2-hydroxybutyric acid, and ß-alanine were isolated. It is tentatively proposed that nitromethane reacts with a dehydroalanine residue at the active site, and that the amino group of the dehydroalanine residue forms a Schiff base to a carbonyl compound that is as yet unidentified.

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