Preparation and reconstitution with divalent metal ions of class I and class II Clostridium histolyticum apocollagenases.
Both gamma- and zeta-collagenases from Clostridium histolyticum are fully and reversibly inhibited by 1,10-phenanthroline at pH 7.5 in the presence of 10 mM CaCl2 with KI values of 0.11 and 0.040 mM, respectively. The inhibition is caused by removal of the single, active-site Zn(II) present in each of these enzymes. The nonchelating analogue 1,5-phenanthroline has no effect on the activity of either enzyme. Dialysis of the enzymes in the presence of 1,10-phenanthroline, followed by back dialysis against buffer containing no chelating agent, gives the respective apocollagenases. Both apoenzymes can be instantaneously and fully reactivated by the addition of 1 equiv of Zn(II). Variable amounts of activity are restored to both apocollagenases by Co(II) and Ni(II) and to gamma-apocollagenase by Cu(II). The activity titration curve for gamma-apocollagenase with Co(II) and Scatchard plots for the reconstitution of gamma-apocollagenase with Cu(II) and Ni(II) and of zeta-apocollagenase with Ni(II) and Co(II) indicate that all activity changes are the result of binding of a single equivalent of these divalent metal ions at the active site of the collagenases. Cd(II) and Hg(II) do not restore measurable activity to either apoenzyme.
Angleton
E L
EL
Department of Chemistry, Florida State University, Tallahassee 32306.
Van Wart
H E
HE
D001051:Apoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001665:Binding Sites; D002413:Cations, Divalent; D003013:Clostridium / Q000201:enzymology*; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D003300:Copper / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators; D007700:Kinetics; D008628:Mercury / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D001051:Apoenzymes; D001059:Apoproteins; D002413:Cations, Divalent; D004793:Enzyme Reactivators; D008670:Metals; D010618:Phenanthrolines; D003035:Cobalt; D003300:Copper; D009532:Nickel; D003012:Microbial Collagenase; D008628:Mercury; D015032:Zinc; C025205:1,10-phenanthroline