Two‐Metal Ion Catalysis in Enzymatic Acyl‐ and Phosphoryl‐Transfer Reactions

Abstract

Numerous studies, both in enzymatic and nonenzymatic catalysis, have been undertaken to understand the way by which metal ions, especially zinc ions, promote the hydrolysis of phosphate ester and amide bonds. Hydrolases containing one metal ion in the active site, termed mononuclear metallohydrolases, such as carboxypeptidase A and thermolysin were among the first enzymes to have their structures unraveled by X-ray crystallography. In recent years an increasing number of metalloenzymes have been identified that use two or more adjacent metal ions in the catalysis of phosphoryl-transfer reactions (R-OPO, + R'-OH + R'-OPO, + R-OH; in the case of the phosphatase reaction R'-OH is a water molecule) and carbonyl-transfer reactions, for example, in peptidases or other amidases. These dinuclear metalloenzymes catalyze a great variety of these reactions, including hydrolytic cleavage of phosphomono-, -di-and -triester bonds, phosphoanhydride bonds as well as of peptide bonds or urea. In addition, the formation of the phosphodiester bond of RNA and DNA by polymerases is catalyzed by a two-metal ion mechanism. A remarkable diversity is also seen in the structures of the active sites of these di-and trinuclear metalloenzymes, even for enzymes that catalyze very similar reactions. The determination of the structure of a substrate, product, stable intermediate, or a reaction coordinate analogue compound bound to an active or inactivated enzyme is a powerful approach to investigate mechanistic details of enzyme action. Such studies have been applied to several of the metalloenzymes reviewed in this article; together with many other biochemical studies they provide a growing body of information on how the two (or more) metal ions cooperate to achieve efficient catalysis.

Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis

“…Upon substrate binding, we predict that the bridging hydroxide becomes terminally bound to Zn 2 while retaining a hydrogen bond to Asp-120. As predicted with model complexes (61), other Zn(II)-and Fe-containing proteins (62)(63)(64)(65), computational studies on metallo-␤-lactamases (40,42,66), and with other metallo-␤-lactamases (57, 67, 68), this metal bound hydroxide then serves as the nucleophile that attacks the ␤-lactam carbonyl. The kinetic data clearly show that D120N is the only mutant that retains significant hydrolytic activity.…”

Cellular function and molecular structure of ecto-nucleotidases

“…In the S102A or S102G variants, the reaction might proceed via a direct attack of a Zn2-coordinated water nucleophile on the substrate phosphate group, similar to mechanisms in other dinuclear metallohydrolases [614]. Interestingly, mutation of R166 (R166Q, R166S, and R166A) increases K m 50-fold but has only minor effects on k cat [615,616].…”

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…In all of the structures, the metals are in distorted octahedral environments, separated by ∼3.5 Å and are bridged by two exogeneous carboxylate groups and the central phenolate ligand ( Figure 7). As an illustration, the crystal structure of [LFe(III)(µ-OAc) 2 …”