Carboxypeptidase. metal chelate enzyme

These enzymes require a metal for activity and are inhibited by metal-chelating compounds. They are exopeptidases and include carboxypeptidase A (peptidyl-L-amino-acid hydrolase) and B (peptidyl-L-lysine hydro-... 

It could be shown, moreover, (Vallee and Neurath, 1955) that five times recrystallized carboxypeptidase was completely inhibited by metal chelating agents, such as 8-OHQ-5SA and 1-10 phenthroline at concentrations of 10 W,Q ,Q Dat concentrations of 10" Af, and some 30% byEDTA at 10 M. These are all known to form complexes with zinc in simple systems. In these experiments, the buffered enzyme solutions were incubated with the chelating agent at pH 7.5, 4°C., for 1 hour prior to the addition of the substrate. Inhibition did not occur when these chelating agents were first incubated with an equimolar amount of zinc, cupric, or ferrous ions. Sodium diethyldithiocarbamate, zincon, sulfanilamide, and diamox, the latter two employed because of their effect on carbonic anhydrase, had little, if any, effect on carboxypeptidase activity. DPN, nicotinamide, and A-methylnicotinamide, examined because of their effect on the ADH sys-... 

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 Recent and unpublished data indicate the time course of the inhibitory effects of these agents OP in concentrations of 1 X 10" M causes 90 % inhibition of the reaction in 60 minutes.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M zinc ions to the enzyme thus inhibited restores enzymatic activity, demonstrating the reversibility of inhibition (unpublished results).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 mechanism of the observed cellular action of lysinoalanine is not well understood (Finot et al., 1977, Finot, 1983 Engelsma et al., 1979 Reyniers, 1979 Leegwater and Tas, 1980). The possible interaction of LAL with metal ions needs to be explored, however, in view of the recent observations both here and by Hayashi (1982) that LAL inhibits the enzymatic activity of metallo-enzymes such as carboxypeptidase, which contains zinc as part of its active site. The inhibition appears reversible since carboxypeptidase activity was regenerated following the addition of zinc sulfate to the LAL-inactivated enzyme. Inhibition is not surprising since LAL contains three amino and two carboxyl groups and structurally resembles ethylenediaminetetraacetic acid (EDTA), a well-known metal chelator. 

An artificial metalloenzyme (26) was designed by Breslow et al. 24). It was the first example of a complete artificial enzyme, having a substrate binding cyclodextrin cavity and a Ni2+ ion-chelated nucleophilic group for catalysis. Metalloenzyme (26) behaves a real catalyst, exhibiting turnover, and enhances the rate of hydrolysis of p-nitrophenyl acetate more than 103 fold. The catalytic group of 26 is a -Ni2+ complex which itself is active toward the substrate 1, but not toward such a substrate having no metal ion affinity at a low catalyst concentration. It is appearent that the metal ion in 26 activates the oximate anion by chelation, but not the substrate directly as believed in carboxypeptidase. 

Carboxypeptidase A was the first zinc enzyme to yield a three-dimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb etai, 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argosetai, 1978 Kesterand Matthews, 1977 Monzingoand Matthews, 1984 Matthews, 1988 Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.. 

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

Metal binding in procarboxypeptidase A is weaker than in the active enzyme ( 107), Table 7). It was proposed that the bonding involves sulfur and a weaker ligand than N (107). In view of the present concept of the chelating site in carboxypeptidase, further studies of the zymogen are necessary. In that connection, the cobalt complex should be valuable. 

Electrophilic action in enzymes is carried out by metal ion or coenzyme. The metal ion which is a component of the active center is bound to imidazole moiety in histidine by chelation. The typical enzymes are carboxypeptidase and decarboxylase, etc. A change in electronic structure of the metal ion by the chelation plays an important role in the catalysis. The catalytic activity of carboxypeptidase can be illustrated as follows (11),... 

Complete removal of zinc and thus inactivation of the enzyme can be accomplished in these systems at low D-PEN concentrations if a secondary scavenger chelator is added to the system. Such chelators bind metal that has been released from the enzyme but do not participate in the release.In the case of carboxypeptidase A, aM thionein (apo-metallothionen see Metallothioneins) inhibits catalysis by only about 10% over a 15-min period consistent with its action as a secondary chelator. However, in the presence of 250 aM D-PEN and aM thionein total inhibition is achieved in less than 15 min. D-PEN accelerates zinc equilibration between carboxypeptidase A and thionein (Scheme 1). This is accomplished by D-PEN catalyzing the release of Zn from the enzyme. Since D-PEN is in vast excess over both the enzyme and thionein, the enzyme-released zinc would be expected to bind to D-PEN first. However, since thionein binds zinc more tightly than D-penicillamine and can accept 7 moles of zinc per mole of thionein, it should be the ultimate acceptor of the released zinc. 

A metal atom is essential to the catalytic activity of carboxypeptidase A (53). The enzyme, as isolated, contains one gram atom of zinc per molecular weight of 34,600. Removal of the metal atom, either by dialysis at low pH or by treatment with chelating agents, gives a totally inactive apoenzyme. Activity can be restored by readdition of zinc or a number of other divalent metal ions (Table VII). The dual activity of carboxypeptidase towards peptides and esters is quite sensitive to the particular activating metal ion. Thus, the cobalt enzyme has twice the activity of the native zinc enzyme toward peptides but the same activity toward esters. Characteristic peptidase and esterase activities are also observed for the and Mn enzymes as well while the Cd ", Rh ", and Pb " en-... 

Recently, Hayashi (92) suggested that the chelating characteristics of LAL could be involved with its toxicity. Since LAL is a strong chelator, LAL could potentially remove metal ions from metaloenzymes, thereby inhibiting the enzyme. Inhibition of carboxypeptidase-B and alcohol dehydrogenase were demonstrated as examples. 

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