Catechol oxidases are ubiquitous plant enzymes containing a dinuclear copper center. In the wound-response mechanism of the plant they catalyze the oxidation of a broad range of ortho-diphenols to the corresponding o-quinones coupled with the reduction of oxygen to water. The crystal structures of the enzyme from sweet potato in the resting dicupric Cu(II)-Cu(II) state, the reduced dicuprous Cu(I)-Cu(I) form, and in complex with the inhibitor phenylthiourea were analyzed. The catalytic copper center is accommodated in a central four-helix-bundle located in a hydrophobic pocket close to the surface. Both metal binding sites are composed of three histidine ligands. His 109, ligated to the CuA site, is covalently linked to Cys 92 by an unusual thioether bond. Based on biochemical, spectroscopic and the presented structural data, a catalytical mechanism is proposed in which one of the oxygen atoms of the diphenolic substrate binds to CuB of the oxygenated enzyme.

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.

No abstract

Kidney bean purple acid phosphatase (KBPAP) is an Fe(III)-Zn(II) metalloenzyme resembling the mammalian Fe(III)-Fe(II) purple acid phosphatases. The structure of the homodimeric 111-kilodalton KBPAP was determined at a resolution of 2.9 angstroms. The enzyme contains two domains in each subunit. The active site is located in the carboxyl-terminal domain at the carboxy end of two sandwiched beta alpha beta alpha beta motifs. The two metal ions are 3.1 angstroms apart and bridged monodentately by Asp164. The iron is further coordinated by Tyr167, His325, and Asp135, and the zinc by His286, His323, and Asn201. The active-site structure is consistent with previous proposals regarding the mechanism of phosphate ester hydrolysis involving nucleophilic attack on the phosphate group by an Fe(III)-coordinated hydroxide ion.

The crystal structure of catechol oxidase reveals new insight into the functional properties of the type-3 copper proteins. This class of proteins includes the closely related and better-known tyrosinase as well as hemocyanin, an oxygen transport protein. All these proteins have a dinuclear copper center, have similar spectroscopic behaviors, and show close evolutionary and functional relationships. Comparison between the 3D structures of catechol oxidase and hemocyanins reveals the structural reasons for the divergence in function.