Kinetic and mutagenic evidence for the role of histidine residues in the Lycopersicon esculentum 1-aminocyclopropane-1-carboxylic acid oxidase

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into th e expression vector PT7-7. The highly expressed protein was recovered in th e form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (D EPC) with a second-order rate constant of 170 M-1 min(-1). The pH-inactivat ion rate data imply the involvement of an amino acid residue with a pK valu e of 6.05. The difference UV spectrum of the the DEPC-inactivated versus na tive ACCO showed a single peak at 242 nm indicating the modification of his tidine residues. The inactivation was reversed by the addition of hydroxyla mine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies in dicate that both iron and ACC bind near the active site, which contains his tidine residues. Four histidines of ACCO were individually mutated to alani ne and glycine. H39A is catalytically active, while H177A, H177G, H211A, H2 11G, H234A, and H234G are basically inactive. The results indicate that his tidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.