The catalytically competent coordination environment of the active site metal ion of liver alcohol dehydrogenase

Abstract

The coordination environment of the active site metal ion of horse liver alcohol dehydrogenase is investigated by EPR and steady-state kinetic methods with use of the native (ZnLADH) and the active site specific Co 2+-reconstituted enzyme (COLADH) described by Zeppezauer and coworkers [1]. The pH dependence of kinetic parameters for the oxidation of benzylalcohol reveals two ionizations (pK 1∼6.7; pK 2∼ 10.6) that govern k cat and belong to the ternary enzyme-NAD +-alcohol complex and two ionizations (pK' 1∼ 7.5; pK' 2∼ 8.9) that govern k cat/K m and belong to the binary enzyme-NAD + complex. Only pK 2 and PK' 2 are substantially influenced by metal substitution. Comparable results are observed for the oxidation of isopropanol. We attribute these ionizations to metal-bound water that occur separately at the ternary and binary complex level in the course of alcohol oxidation. In parallel studies from this laboratory [2, 3], we have demonstrated that the magnitude of the zero-field splitting (ZFS) on the high-spin Co 2+ ion falls into three ranges according to coordination number: 0–13 cm −1 for tetracoordinate; 20–60 cm −1 for penta-coordinate; and 90–310 cm −1 for hexa-coordinate environments. We have determined the ZFS energy (2 D) of the Co 2+ ion in a variety of binary and ternary complexes of CoLADH to assign the coordinates number of the active site metal ion. The results are 9.3 cm −1 (CoLADH); 3.1 cm −1 (CoLADH-CF 3CH 2OH); 13 cm −1 (CoLADHNADH.N,N-dimethylaminocinnamaldehyde); and 8.3 cm −1 (CoLADH-tetrahydroNADH), indicative of tetracoordinate sites, while the ZFS constants of the CoLADH-NADH, CoLADH-NADH-benzylalcohol, CoLADH-NADH-CF 3CH 2OH, and CoLADH-NAD +CF 3CH 2OH complexes are >20 cm −1, indicative of pentacoordinate environments. The results taken together indicate that the active site metal ion is pentacoordinate in catalytically component reaction intermediates and is ligated by a neutral water molecule in the physiologically active ternary enzymeNAD +alcohol complex. We suggest that the neutral metal-bound water molecule serves as the base catalyst for abstraction of the proton from the alcoholic hydroxyl group of the substrate. We present a mechanism for the catalytic action of LADH consistent with these observations and indicate how the metal-bound water molecule may modulate the Lewis acid reactivity of the active site metal to control the catalytic action of LADH. (Supported by NIH grant GM 21900).