Catalytically active metalloenzyme

Metallocene (Section 14 14) A transition metal complex that bears a cyclopentadienyl ligand Metalloenzyme (Section 27 20) An enzyme in which a metal ion at the active site contributes in a chemically significant way to the catalytic activity... 

Several model systems related to metalloenzymes such as carboxypeptidase and carbonic anhydrase have been reviewed. Breslow contributed a great deal to this field. He showed how to design precise geometries of bis- or trisimidazole derivatives as in natural enzymes. He was able to synthesize a modified cyclodextrin having both a catalytic metal ion moiety and a substrate binding cavity (26). Murakami prepared a novel macrocyclic bisimidazole compound which has also a substrate binding cavity and imidazole ligands for metal ion complexation. Yet the catalytic activities of these model systems are by no means enzymic. 

Metalloenzymes with non-heme di-iron centers in which the two irons are bridged by an oxide (or a hydroxide) and carboxylate ligands (glutamate or aspartate) constitute an important class of enzymes. Two of these enzymes, methane monooxygenase (MMO) and ribonucleotide reductase (RNR) have very similar di-iron active sites, located in the subunits MMOH and R2 respectively. Despite their structural similarity, these metal centers catalyze very different chemical reactions. We have studied the enzymatic mechanisms of these enzymes to understand what determines their catalytic activity [24, 25, 39-41]. 

Metal-dependent enzymes have been divided into two groups,80-83 the metalloenzymes and the enzyme-metal-ion complexes. Metalloenzymes are those that contain one or more functional metal atoms per enzyme molecule. The metal is firmly bound to the protein, and the enzyme can be purified without any loss in activity. The content of functional metal in the preparation approaches a limiting value during purification. Enzyme-metal-ion complexes are more readily dissociable than metalloenzymes. It is necessary to add the functional, metal ion during or after purification, in order to maintain or restore full catalytic activity. 

As the Zn2+ required for catalytic activity could be retained during purification, a-D-mannosidase may be classified as a metalloenzyme. The enzyme seems to be completely specific for Zn2+ as the activating cation. 

Figure .17 Artificial metalloenzymes (a) Strategyto incorporate a catalytically active metal fragment within a host protein.

Zinc in metalloenzymes may (i) participate directly in the catalytic process, (ii) serve to stabilize protein structure or (iii) have a regulatory role. In each case, removal of the metal from the holoenzyme generally results in an apoprotein having no catalytic activity. The enzymes considered briefly below provide examples of each of these functions of Zn. The study of zinc metalloproteins has often in the past been beset by analytical problems and by contamination with traces of metal ions a review covering these important topics has appeared.1263 Another recent review deals with the physiological, nutritional and medical role of zinc.1264... 

In zinc metalloenzymes. zinc is a selective stoichiometric constituent and is essential for catalytic activity. It is frequently present in numerical correspondence with the number of active enzymatic sites, coenzyme binding sites, or enzyme subunits Removal of zinc results in loss of activity. Inhibition by metal complexing agents is a characteristic feature of zinc metalloenzymes. However, no direct relationship holds between the inhibitory effectiveness of these agents and their affinity for ionic zinc. Although zinc is the only constituent of zinc metalloenzymes in vivo, it can be replaced by other metals m vitro, such as cobalt, nickel, iron, manganese, cadmium, mercury, and lead, as m the case of carboxy-peprida.ses. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

In a number of cases, a common pattern for catalytic activity seems to be developing, where the zinc centre, already four-coordinate with three protein ligands and an aqua group, binds the substrate to give a five-coordinate complex. The zinc-bound aqua group is ionized to give hydroxide, which participates in the catalytic reaction. Changes in coordination number of the zinc from four to five and back to four appear to be a common feature of zinc metalloenzymes. 

Alkaline phosphatase160-164 is a dimeric zinc metalloenzym composed of two identical subunits. The number of zinc atoms per protein molecule varies in different preparations. However, only two seem to be required for catalytic activity. The molecular weight of the monomer has been reported to be 42.000 so the natural dimer would be twice that value. Alkaline phosphatase is a phosphorylating enzyme and has 760 residues per dimer. 

Metal-catalysed hydrolysis of / -nitrophenyl picolinate at pH 7.5 was in the order Cu(II) > Ni(II) > Zn(II) > Co(II) > La(III). The probable mechanism is via attack by external HO- on the metal-ion complex (80).80 High catalytic activity in the hydrolysis at pH 7 of p-nitrophenyl picolinate, but not / -nitrophenyl acetate, was displayed by the metal complexes M(2-aminopyridine)2(OAc)2 (M = Zn, Ni), showing that they were good models for hydrolytic metalloenzymes.81... 

Roughly 30% of enzymes are metalloenzymes or require metal ions for activity and the present chapter will concentrate on the chemisty and structure of the plant metalloenzymes. As analytical methods have improved it has been possible to establish a metal ion requirement for a variety of enzymes which were initially considered to be pure proteins. A dramatic example is provided by the enzyme urease isolated from Jack beans and first crystallised by Sumner (1926) (the first enzyme to be crystallised). Sumner defined an enzyme as a pure protein with catalytic activity, however, Zerner and his coworkers (Dixon et al., 1975) established that urease is in fact a nickel metalloenzyme. Jack bean urease contains two moles of nickel(II) per mole of active sites and at least one of these metal ions is implicated in its mechanism of action. 

Photosynthesis can be affected in many ways. Metals can influence biosynthesis of biomembranes and photosynthetic pigments, especially chlorophyll. They may inactivate enzymes by oxidising SH-groups necessary for catalytic activity or by substitution for other divalent cations in metalloenzymes. They finally can also interact with the photosynthetic electron transport and with the related photophosphorylation. 

Metal ions are vital to the function of many enzymes that catalyze hydrolytic reactions. Coordination of a water molecule to a metal ion alters its acid-base properties, usually making it easier to deprotonate, which can offer a ready means for catalyzing a hydrolytic reaction. Also, the placement of a metal center in the active site of a hydrolytic enzyme could permit efficient delivery of a catalytic water molecule to the hydrolyzable substrate. In fact, the first enzyme discovered, carbonic an-hydrase, is a metalloenzyme that requires a Zn2+ center for its catalytic activity (32). The function of carbonic anhydrase is to catalyze the hydrolysis of carbon dioxide to bicarbonate ... 

FIGURE 28 Artificial metalloenzymes (A) strategy for incorporating a catalytically active metal fragment into a host protein (Wilson and Whitesides (97)) (B) hydrogenation of alkenes via biotin-(strep)avidin methodology (Wilson and Whitesides (97) and Skander et al. (9S)). (For a color version of this figure, the reader is referred to the Web version of this chapter.)... 

Traditionally, when the association between the metal ion and the protein is relatively strong (i.e., binding constant higher than 10 the complex is called a metalloprotein. When the protein is performing catalytic activity at the metal center, it is called a metalloenzyme. 

The biological catalytic activity of metalloproteins for redox reactions is usually associated with a particular coordination environment of the metal active site [160, 161], In particular, there has been considerable interest in 02-binding and -activation by non-heme metalloenzymes [162-167). A redox-active metal center is often associated with another metal center which can accelerate the redox process of O2... 

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