Soluble and immobilized clostridial aminopeptidase and aminopeptidase P as metal-requiring enzymes.
The dependence of enzymatic activity on Co2+ concentration was found to be bell-shaped for the soluble and immobilized clostridial aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.13) and aminopeptidase P (aminoacylpropyl-peptide hydrolase, EC 3.4.11.9), with maxima in the 3-18 microM range of Co2+ concentration. The Co2+-enzyme association constants derived from the activation of soluble, glass- and cellulose-bound clostridial aminopeptidase by Co2+ were KE-Co = 5.2 X 10(5), 4.5 X 10(6) and 2.0 X 10(5) M-1, respectively; for soluble and glass-bound aminopeptidase P, the KE-Co were 1.5 X 10(5) and 8.2 X 10(5) M-1, respectively. Kinetic measurements indicate the involvement of Co2+ in the enzyme-substrate binding. Cobalt-citrate (Co-cit) acted as a useful metallobuffer and protected both enzymes against inhibition by high concentrations of CoSO4. For association of citrate with Co2+ under the assay conditions, KCo-cit was determined as (5.3 +/- 1.4) X 10(3) M-1 by anodic stripping polarography. In contrast to the rapid association of Co2+ with soluble and glass-bound clostridial aminopeptidase (less than 1 min at 4 degrees C), the dissociation process was very slow (hours to days), being slower for the glass-bound than for the soluble and cellulose-bound enzyme. For aminopeptidase P, both processes were rapid. All the interactions were shown to be reversible.
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002413:Cations, Divalent; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D003013:Clostridium / Q000201:enzymology*; D003035:Cobalt / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000378:metabolism; D007700:Kinetics; D011048:Polarography
D002413:Cations, Divalent; D002951:Citrates; D004800:Enzymes, Immobilized; D019343:Citric Acid; D003035:Cobalt; D000626:Aminopeptidases; C043040:clostridial aminopeptidase; C022750:X-Pro aminopeptidase