Mechanistic Studies of 1-Aminocyclopropane-1-carboxylate Deaminase: Characterization of an Unusual Pyridoxal 5′-Phosphate-Dependent Reaction

摘要

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 50n-phosphaten(PLP)-dependent enzyme that cleaves the cyclopropane ring ofACC, to give R-ketobutyric acid and ammonianas products. The cleavage of the CR-Cβ bond of an amino acid substrate is a rare event in PLP-dependentnenzyme catalysis. Potential chemical mechanisms involving nucleophile- or acid-catalyzed cyclopropane ringnopening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode ofncyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features ofnACCDcatalysis by investigating the kinetic properties ofACCDfromPseudomonas sp.ACP and several of itsnmutant enzymes. Our studies suggest that the pKa of the conserved active site residue, Tyr294, is lowered by anhydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonatenthe incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLPncoenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave thencyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and 13nCnKIE studies of the wild type enzyme suggest that the CR-Cβ bond cleavage step in the chemical mechanism isnat least partially rate-limiting under kcat/Km conditions and is likely preceded in the mechanism by a partiallynrate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive externalnaldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context ofnpreviousmechanistic and structural studies ofACCDenzymes, our studies aremost consistent with amode ofncyclopropane ring cleavage involving nucleophilic catalysis by Tyr294.