Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism

Histidine ammonia-lyase (EC 4.3.1.3) catalyzes the non-oxidative elimination of the alpha-amino group of histidine using a 4-methylidene-imidazole-5-one (MIO). which is formed autocatalytically from the internal peptide segment 142Ala-Ser-Gly. The structure of the enzyme inhibited by a reaction with L-cysteine was established at the very high resolution of 1.0 Angstrom. Five active center mutants were produced and their catalytic activities were measured. Among them, mutant Tyr280 --> Phe could be crystallized and its structure could be determined at 1.7 Angstrom resolution. It contains a planar sp(2)-hybridized 144-N atom of MIO, in contrast to the pyramidal sp(3)-hybridized 144-N of the wild-type. With the planar 144-N atom, MIO assumes the conformation of a putative intermediate aromatic state of the reaction, demonstrating that the conformational barrier between aromatic and wild-type states is very low. The data led to a new proposal for the geometry for the catalyzed reaction, which also applies to the closely related phenylalanine ammonia-lyase (EC 4.3.1.5). Moreover, it suggested an intermediate binding site for the released ammonia.