Thiol-group binding of zinc to a beta-lactamase of Bacillus cereus: differential effects on enzyme activity with penicillin and cephalosporins as substrates.
Zinc, which is required for the hydrolysis of cephalosporins by a crude enzyme from Bacillus cereus 569, also increased the stability of this activity during storage. A loss in activity of the zinc-activated enzyme which occurred on prolonged hydrolysis of cephalosporin C was not restored by further addition of zinc. The thiol reagents N-ethyl maleimide (NEM), iodoacetic acid (IAA), CdCl(2), and p-chloromercuribenzoate, all at 10(-3)m, and iodine at 1.6 x 10(-3)n prevent zinc activation of the "cephalosporinase" activity. However, NEM and IAA have minimal or no demonstrable inhibitory effect if the enzyme is first treated with zinc. This suggests that zinc is linked to the apoenzyme by a thiol group. Activation by zinc is only partially prevented by NEM if the crude enzyme is pretreated with nickel, which alone causes negligible activation of the apoenzyme. The order of affinities of these metals for the apparent thiol group is thus Hg(++), Cd(++) > Zn(++) > Ni(++). The "cephalosporinase" inhibition by Hg(++) was reversible with dithiothreitol. These metals and thiol reagents do not decrease the ability of the crude enzyme to hydrolyze benzylpenicillin, which is consistent with the report that purified "penicillinase" from B. cereus contains no cysteine residue. This suggests that the beta-lactamases of B. cereus that hydrolyze penicillin and cephalosporins differ from each other by at least one amino acid (cysteine).
D000327:Adsorption; D001409:Bacillus cereus / Q000187:drug effects / Q000201:enzymology*; D001426:Bacterial Proteins / Q000032:analysis; D002104:Cadmium / Q000494:pharmacology; D002511:Cephalosporins / Q000378:metabolism*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002845:Chromatography; D003956:Dialysis; D004790:Enzyme Induction; D005033:Ethylmaleimide / Q000494:pharmacology; D007455:Iodine / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D010405:Penicillinase / Q000032:analysis / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology*
D001426:Bacterial Proteins; D002511:Cephalosporins; D002729:Chloromercuribenzoates; D007461:Iodoacetates; D010406:Penicillins; D002104:Cadmium; D009532:Nickel; D007455:Iodine; D010405:Penicillinase; D008628:Mercury; D015032:Zinc; D005033:Ethylmaleimide