Metal inhibitor-chelate structure

The first enzyme that was demonstrated to contain nickel was urease (urea amidohydrolase) from jack bean. It catalyzes the hydrolysis of urea to ammonia and carbon dioxide. The protein has a multimeric structure with a relative molecular mass of 590,000 Da. Analysis indicated 12 nickel atoms/mol. Binding studies with the inhibitors indicated an equivalent weight per active site of 105,000, corresponding to 2 nickel atoms/active site. During removal of the metal by treatment with EDTA at pH 3.7, the optical absorption and enzymatic activity correlated with nickel content. This, combined with the sensitivity of the enzyme to the chelating agents acetohydroxamic acid and phos-phoramidate, indicates that nickel is essential to the activity of the enzyme (1). 

Another set of weak Icmt inhibitors from natural sources were also discovered by Buchanan and coworkers [75]. Prenylated p-hydroxy chal-cone derivatives along with the known flavone, (5)-glabrol, were discovered from the extract Hovea parvicalyx and found to possess Icmt inhibitory activity with an IC50 of 17 pM. Interesting to note that the p-hydroxychal-cones have a structural resemblance to some metal chelators. Continued studies on these natural products will further give the medicinal chemist insight into the continued refinement of Icmt inhibitors. 

The effects of increasing concentrations of 8-OHQ-5SA, OP, and aa D on the activity of carboxypeptidase at a constant substrate concentration of 0.02 M CGP are shown in Fig. 2. (Vallee and Neurath, 1955.) Activity of the inhibited reaction was expressed as per cent of the proteolytic coefficient observed at zero inhibitor concentration. The conditions of preincubation are indicated. Recent and unpublished data indicate the time course of the inhibitory effects of these agents OP in concentrations of 1 X 10" M causes 90 % inhibition of the reaction in 60 minutes. 80 % of the inhibition occurs in the first 15 minutes (Fig. 3), Addition of 1 X 10" M zinc ions to the enzyme thus inhibited restores enzymatic activity, demonstrating the reversibility of inhibition (unpublished results). Since inhibition did not occur when chelating agents were first incubated with zinc, cupric, or ferrous ions to form the respective metal chelate, it appeared that the sites of chelation of these compounds are responsible for the observed inhibition. Inhibition is therefore not caused by any structural similarity between the inhibitors and the substrate. 

The binding of 1,10-phenanthroline has been studied by X-ray methods (112) in order to correlate a large number of inhibitor studies in solution with the structure (Section II,H,l,b). This chelating agent binds to the zinc atom in the active site displacing the water molecule. The metal is, thus, five coordinated in the complex. The aromatic ring system of the inhibitor is positioned in the active site pocket partly overlapping the assumed nicotinamide position. 

It is an unexpected result from the numerous inhibitor studies summarized here to see the iron (or other metal) ribonucleotide reductases so little impressed by the classical, metal chelating ability of these compounds. It is the unique free radical structure of the metalloenzymes that makes the proteins vulnerable to inactivation while the metals themselves appear much less attractive or accessible as target. 

Most of die binding site residues are conserved in the plant and rat enzymes and the x-ray structures of the different enzyme-inhibitor complexes are very similar, with little movement of the protein backbone (Figure 3). In the apo structure, two histidines, a glutamic acid and three water molecules coordinate the active site metal ion. In the inhibitor bound structures, the 1,3-diketone of the inhibitor chelates the metal ion, replacing two of the water molecules. There... 

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