Zinc ion in enzymes

The most important chemical function of Zn2+ in enzymes is probably that of a Lewis acid providing a concentrated center of positive charge at a nucleophilic site on the substrate/ This role for Zn2+ is discussed for carboxypeptidases (Fig.12-16) and thermolysin, alkaline phosphatase (Fig. 12-23),h RNA polymerases, DNA polymerases, carbonic anhydrase (Fig. 13-1),1 class II aldolases (Fig. 13-7), some alcohol dehydrogenases (Fig. 15-5), and superoxide dismutases (Fig.16-22). Zinc ions in enzymes can often be replaced by Mn2+, Co2+, and other ions with substantial retention of catalytic activity/ ... 

The functioning of zinc ions in enzymes has been controversial and other mechanisms have been proposed. Makinen et al. suggested a transient pentacoordinate Zn2+ complex on the basis of EPR measurements on en-... 

The small molecule examples discussed in this review provide a rich collection of data pertaining to the catalytic functions which zinc ion can perform as an electrophile in aqueous solution. The high resolution structural information on the zinc-metalloproteins together with information derived from chemical modifications, kinetic studies, and other spectroscopic techniques place severe constraints on the possible roles played by zinc ion in enzyme catalytic mechanism. As a consequence, the focus of this review is placed on the relationship of these... 

The literature relevant to model systems involving electrophilic catalysis by divalent metal ions suggests the following catalytic functions for the involvement of zinc ion in enzymic catalysis ... 

The general physical and chemical properties of several of the zinc-metalloenzymes discussed here have been the subjects of excellent comprehensive reviews by Coleman (30), Lindskog et al. (31), Lindskog (32), Hartsuck and Lipscomb (33), Quiocho and Lipscomb (34), and Sund and Theorell (35). Therefore, it would serve no purpose to attempt a comprehensive review of zinc-metalloenzymes. For this reason, the present work is confined primaril to a review of the literature which pertains to the role(s) played by zinc ion in enzyme catal5dic mechanism. This work is further restricted by limiting discussion to consideration of only those enzyme systems for which high resolution 3-dimensional X-ray structures are available. 

It is of interest to note that all members of the endopeptidase family sequenced to date share a consensus site (-His-Glu-X-X-His-) for the binding of the zinc ion in the active site (21). Typically the metalloproteases are found with only the zinc ion bound to the consensus site however, in vitro several other divalent cations can replace the zinc ion (Co+2 often is able to restore native enzyme activity levels)(22,25). In certain cases the metal ion has also been shown to play a structural role as well as a catalytic role (24). 

Although a-D-mannosidase from mammalian, plant, and molluscan sources is dependent upon zinc for its catalytic activity, the addition of this ion has a marked effect in the enzyme assay only at those pH values where the active, protein-metal complex dissociates appreciably despite the presence of substrate. (Dissociation, which is greater at lower values of pH, is lessened in the presence of substrate.) The presence of zinc ion in the assay (0.1 mM) is thus of particular importance in the case of the limpet enzyme, where the pH of optimal activity is 3.5. Jack-bean and rat-epididymal a-D-mannosidase are both assayed at pH 5, and up to 10% activation may be observed with zinc. 

The function of zinc ions may be either catalytic or stractural. Enzymes with a co-catalytic center of two or even three zinc ions in close proximity are also known. In a new type of zinc-binding site, the protein interface, zinc ions are fixed at the interface of two proteins with the aid of amino acid residues. The ligand residues are usually His, Asp, Glu or Cys, which interact via nitrogen, oxygen or sulfur donors with the metal ion. In catalytic binding sites. His coordination dominates and an additional reactive water molecule is bound. 

Both famesyltransferases70 761 and geranylgeranyl-transferases72/77 78b have been characterized, and the three- dimensional structure of the former has been established.73 75-76 The two-domain protein contains a seven-helix crescent-shaped hairpin domain and an a,a-barrel similar to that in Fig. 2-29. A bound zinc ion in the active site may bind the -S group of the substrate protein after the famesyl diphosphate has been bound into the active site.76 79 These enzymes are thought to function by a carbocation mechanism as shown in Eq. 22-3 and with the indicated inversion of configuration.71... 

It was originally assumed that in LADH the zinc existed in an octahedral form with six bonds available for coordination, until in 1967 Vallee and co-workers showed that the enzyme contained two different types of zinc atom.13773 Loss of two zinc atoms from the enzyme resulted in loss of catalytic activity but maintained the tertiary structure. It was postulated from this that one metal ion per subunit played a role maintaining the tertiary structure, while the other zinc functioned in a catalytic role. Only two of the zinc ions in the liver enzyme interact with the inhibitors 1,10-phenanthroline and 2,2 -bipyridyl, thus demonstrating the different chemical reactivities of the zinc ions.1378 It was also shown that one zinc per subunit could be selectively exchanged or removed by dialysis. This modified enzyme containing one zinc per subunit did not bind 1,10-phenanthroline, hence the catalytic zinc is removed first during dialysis.1379 The second zinc atom can be selectively removed in preference to the catalytic zinc, by carboxymethylation followed by dialysis.1377 ... 

The zinc ion in a neutral protease from Bacillus subtilis has been exchanged with other metal ions (139—141). The Co(II) enzyme is reported to be active (140). 

The increase in the amount of copper and decrease in the amount of zinc removed by activated sludge, as the residence time is increased, can be explained by transport processes, adsorption onto surfaces orthe possible competitionbetween copper and zinc ions in binding to available sites on the enzymes. It may be that the interaction between copper and biomass increases as the residence time increases eventually leading to an increase in the amount of copper taken up by activated sludge. As a result, the affinity of zinc for the... 

Some insight into the function of the zinc ion in alkaline phosphatase from E. coli has resulted from 35C1 n.m.r. studies.229 The uncertainty which has surrounded the number of zinc ions required for activity of the enzyme has been resolved somewhat by the observation that alkaline phosphatase prepared in the absence of edta requires only 2 moles of Zn2+ per mole of enzyme for full activity. 35C1 line-broadening by n.m.r. shows that on addition of two moles of zinc per mole enzyme, no broadening... 

The metalloproteases (MPs) and matrix metalloproteinases (MMPs) are a class of metallohydrolases of particular interest to the pharmaceutical industry due to their role in a number of pathological processes [81-83], The lack of an enzyme-bound nucleophilic residue in the metallohydrolases complicates the design of ABPP probes for this class of enzymes. Rather than mechanism-based and electrophilic probes for ABPP, photoreactive variants of reversible inhibitors of metallohydrolases have been developed [84-86]. These reversible inhibitors usually contain a hydroxamate moiety that is capable of chelating the catalytic zinc ion in a bidentate manner [79, 80]. The hydroxamate moiety was incorporated into the first generation of metallohydrolase ABPP probes along with a benzophenone group capable of covalent bond formation upon UV irradiation (Scheme 4). 

Generally speaking, the role of the enzyme consists of the selective and specific attraction of substrate and the highly efficient catalysis. Every enzyme has its own characteristic feature for example, the specificity in the binding and a charge-relay action in the catalysis in a-chymotrypsin, the contribution of the imidazole moiety as an electron donor to the electrophilicity of zinc ion in carboxypeptidase, the change in the spin state and the reactivity of the transition metal ion by the coordination of the imidazole in the hemochrome. These typical characteristic features are the result of the cooperative actions of the constituents. 

Zinc metalloproteases see Zinc Enzymes) form an important class, distinguished by a strict requirement for a tightly bound mononuclear zinc ion cofactor. The role of the zinc ion in the hydrolytic mechanism is to polarize and activate both the coordinated peptide carbonyl and a coordinated solvent nucleophile, stabilizing the tetrahedral intermediate... 

Matrix metalloproteases (MMP) are also inhibited by hydroxamic acids and/or thiols. Over 25 variants of these enzymes are known, and some are involved in diseases ranging from inflammation to metastatic cancer (108). MMPs contain a zinc ion in the active site and function through the metallopeptidases catalytic mechanism already discussed. However, subtle differences between enzymes enable selective inhibitors to be developed (109). Fig. 15.25 lists some of the reported MMP inhibitors that use carboxylic acid (52-53), a hydroxamic acid (54-55), or thiol groups (56)as metal chelators. 

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