Catalysis by Zn 2+-metalloenzymes proceeds via a penta-coordinate metal-OH 2 complex

Abstract

The active site of a variety of metalloenzymes contains a catalytically essential tetra-coordinate Zn 2+ ion to which a water molecule is a ligand. It is generally assumed that the metal-bound water molecule has no role in catalysis. However, for both the native Zn 2+- and Co 2+-reconstituted forms of carboxypeptidase A (CPA) and liver alcohol dehydrogenase (LADH), we have identified metal ion dependent ionizations that govern steady state kinetic parameters and can be ascribed only to a metal-bond water molecule in catalytically competent reaction intermediates [1, 2]. To assign the coordination environment of the Co 2+ ion in these reaction intermediates, we have identified spectroscopic parameters of the high-spin Co 2+ ion that serve as a direct index of coordination number. Our studies [3] show on the basis of a group theoretical treatment of spin-orbit coupling, together with relationships derived through second-order perturbation theory, that the magnitude of the zero-field splitting (ZFS) of the high-spin Co 2+ ion is a direct monitor of crystal field symmetry and that it follows the order ZFS 4 < ZFS 5 < ZFS 6 where the subscript indicates the coordination number. Measurement of the ZFS in structurally defined coordination complexes confirms this relationship and indicates that the magnitude of 2| D| is 0–13 cm −1 for tetra-coordinate complexes, 20–60 cm 1̄ for penta-coordinate complexes, and 90–130 cm −1 for hexa-coordinate complexes. Application of this theory to measurements of the ZFS of CoCPA and CoLADH shows that the active site metal ion is tetra-coordinate in the free enzyme and is altered to a penta-coordinate complex in true reaction intermediates. In these intermediates the metal ion coordinates both the substrate and a water molecule simultaneously. Together with kinetic studies identifying ionizations of a metal-bound water molecule and showing that the reaction pathway is identical for Zn 2+- and Co 2+-enzymes, the results require that, upon formation of a penta-coordinate Zn 2+OH 2 complex, breakdown of the acyl-enzyme (mixed anhydride) reaction intermediate of CPA occurs via metal-hydroxide nucleophilic attack on the carbonyl carbon of the scissile bond while in the physiologically active, ternary enzyme-NAD +- alcohol complex of LADH a neutral metal-bound water molecule serves as the base catalyst for proton abstraction. The results of these studies show that the metal-bound water has an integral catalytic role in CPA and LADH. It is probable that the metal-OH 2 complex has similarly a functional role in other Zn 2+- metalloenzymes. (Supported by NIH grant GM 21900).