Structure−Activity Studies of l-Canaline-Mediated Inhibition of Porcine Alanine Aminotransferase

l-Canaline [l-2-amino-4-(aminooxy)butanoic acid] (l-CAN) and a family of eleven structurally related analogs were synthesized and evaluated for their inhibitory effect on PLP-dependent alanine aminotransferase (AlaAT) (EC 2.6.1.2) obtained from porcine heart. These congeners were selected to determine the stereochemical, aliphatic chain length, and aminooxy substitutional effects on l-CAN-mediated inhibition of AlaAT activity. l-CAN was the most effective inhibitor of the tested compounds; 10-7 M l-CAN elicited a 55% reduction in AlaAT activity after a 5 min exposure. This deleterious effect results from the ability of l-CAN to react avidly with the PLP moiety of the enzyme to form a stable, l-CAN-PLP oxime. In contrast, the methyl and ethyl esters of l-CAN reduced AlaAT activity by only 8% and 6%, respectively. While all of the l-enantiomeric forms of the tested compound were more potent AlaAT inhibitors than their corresponding d-stereoisomers, the d-enantiomers, particularly d-canaline, were active. Chain shortening or lengthening dramatically curtailed l-CAN-mediated loss in AlaAT activity, but the replacement of the α-amino group with a hydrogen was of little consequence in this regard. AlaAT was treated with l-CAN in the presence of free PLP to assess PLP capacity to protect AlaAT against 10-7 M l-CAN-dependent inactivation. l-CAN retained approximately two-thirds of its inhibitory ability in the presence of equimolar PLP, but AlaAT inhibition was reduced 90% by a 10-fold excess of PLP over l-CAN.