Metal cofactor requirements of beta-lactamase II.
1. The apoenzyme obtained on removal of Zn(2+) from beta-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn(2+)-containing enzyme. 2. Removal of Zn(2+) led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn(2+) by Co(2+), Cd(2+), Mn(2+) or Hg(2+) gave enzymes with significant, but lower, beta-lactamase activity. No activity was detected in the presence of Cu(2+), Ni(2+), Mg(2+) or Ca(2+). 4. Equilibrium dialysis indicated that the enzyme had at least two Zn(2+) binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn(2+) indicated that activity paralleled binding of Zn(2+) to the site of highest affinity. 5. Replacement of Zn(2+) by Co(2+) and Cd(2+) gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured beta-lactamase II with Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce beta-lactamase II activity in high yield.
D001409:Bacillus cereus; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium; D002250:Carbon Radioisotopes; D002511:Cephalosporins; D002942:Circular Dichroism; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004789:Enzyme Activation; D006868:Hydrolysis; D066298:In Vitro Techniques; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D008667:Metalloproteins; D009532:Nickel / Q000494:pharmacology; D010405:Penicillinase / Q000032:analysis; D011485:Protein Binding; D011489:Protein Denaturation; D011868:Radioisotopes; D012588:Scintillation Counting; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000494:pharmacology; D015033:Zinc Isotopes
D002250:Carbon Radioisotopes; D002511:Cephalosporins; D008024:Ligands; D008667:Metalloproteins; D011868:Radioisotopes; D015033:Zinc Isotopes; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D003300:Copper; D009532:Nickel; D010405:Penicillinase; D008628:Mercury; D008274:Magnesium; D015032:Zinc; D002118:Calcium