Metalloenzymes inhibition

The analysis of such inhibition curves has been based on the implied assumption that these metal chelate complexes (E. L. Smith and Hanson, 1949) are analogous to those observed in simple systems (Martell and Calvin, 1952), an assumption for which little experimental justification exists. It should be emphasized that a metalloenzyme inhibited by binding of a chelating agent to a metal in situ represents a mixed complex, consisting of two different ligands combined with one metal. Studies of the physical chemistry of simple complexes of this type are as yet few (Klotz and Loh-Ming, 1954 de Witt and Watters, 1954). The data on mixed complexes available to date are apparently not adequate to justify extrapolations to the interpretation of inhibition of metalloenzymes (Coryell, 1955, personal communication). 

Cyanide can inhibit enzymatic activity by binding to the metallic cofactor in metalloenzymes. 

Hydroxamic acids are important bioligands and are involved in numerous biological processes including metal-ion transport and inhibition of metalloenzymes . 1 1 Metal binding to hydroxamic acids usually occurs in a bidentate fashion (Scheme 99) with... 

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. 

Metal-binding agents (118-120) Oxalate, citrate, 1,10-phenanthroline, azide, diethyldithiocarbamate, cyanide, and 8-hydroxyquinoline inhibit, suggesting the metalloenzyme nature of the catalyst... 

Independently, simple peptide hydroxamic acids (Z-Gly-L-Leu-NHOH and others) were first observed to inhibit the metalloprotease thermolysin in 1977 9,101 The structure was then further improved to the hydroxamidoalkylmalonyl-peptide moiety by considering the substrate specificity of thermolysin and other metalloproteases 10-121 A summary of hydroxamic acids reported to be inhibitors of various metalloenzymes up to 1983 has been published 131 In 1985 hydroxamido-benzylsuccinyl-L-alanine (kelatorphan) was synthesized and found to be one of the best enkephalinase inhibitors 141... 

Fig. 7.2 Tlie crystal structure of mammalian Ser/Thr protein phosphatase-1, complexed with the toxin mycrocystin was determined at 2.1 A resolution. PPl has a single domain with a fold, distinct from that of the protein tyrosine phosphatases. The Ser/Thr protein phosphatase-1, is a metalloenzyme with two metal ions positioned at the active site with the help of a p-a-p-o-p scaffold. A dinuclear ion centre consisting of Mn2+ And Fe2+ g situated at the catalytic site that binds the phosphate moiety of the substrate. Ser/Thr phosphatases, PPl and PP2A, are inhibited by the membrane-permeable ocadaic acid and by cyclic hexapeptides, known as microcystins. The toxin molecule is depicted as a ball-and-stick structure. On the left and on the ri t, two different views of the same molecule are shown. Microcystin binds to three distinct regions of the phosphatase to the metaLbinding site, to a hydrophobic groove, and to the edge of a C-terminal groove in the vicinity of the active site. At the surface are binding sites for substrates and inhibitors. These ribbon models are reproduced vnth permission of the authors and Nature from ref. 9.

Since the zinc-bound water is one of the most critical components of the catalytic zinc site, there are three ways that one can envision inhibiting the metalloenzyme. Thus inhibition should occur if a potential inhibitor can displace the water by binding directly to the zinc, or remove the zinc ion from the protein or bind a second zinc ion to the catalytic zinc-bound water and/or another catalytic group (Figure 8). 

Figure 8 Inhibition of zinc metalloenzymes is accomplished by (a) an Inhibitor displacing the zinc-bound water, (b) removing the metal, or (c) bridgiag an inhibitory zinc to the catal)dic zinc...

Investigations into the metalloenzyme nature of an enzyme frequently involves an attempt to inhibit it with a chelator, followed by reversal of any inhibition by adding excess metal. If excess zinc doesn t reverse the inhibition, it may be due to the fact that zinc can inhibit the zinc enzyme. A considerable amount of anecdotal information exists that zinc inhibits zinc enzymes but how it accomplishes it is not well established. The most thorough study of its mechanism of inhibition is on CPD... 

A few reports on metal ion characterization in metalloenzymes by hyphenated techniques with ICP-MS detection should also be mentioned. Inhibition, reactivation, and determination of metal ions in membrane metalloproteases of bacterial origin was studied by Leopold et al The results obtained by SEC-ICP-MS and the results of enzymological methods indicated that two different membrane proteases from Bacillus cereus and Pseudomonas aeruginosa were zinc metalloproteases. In another study, ICP-MS measurements helped in the characterization of a fibrinolytic metalloprotease from the ftniting bodies of an edible mushroom. " Using HPLC-ICP-MS, Suzuki et al. detected a zinc-binding protein present specifically in the livers of male adult rats. ... 

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