Inhibition of cobalt(II) carboxypeptidase A by ligands. Kinetic evidence for the formation of a ternary enzyme-metal-ligand complex.

Saturation kinetics are observed in the inhibition of cobalt carboxypeptidase A by the chelating agent 1,10-phenanthroline. The association constant K1 for the formation of the enzyme-metal-ligand ternary complex and k2, the rate of breakup of the ternary complex, have been obtained. A mechanism is proposed to account for the pH profile of the reaction which, in conjunction with K1, permits the calculation of the individual rate constants k1, k-1, k2, k3. The magnitude of the rate constant k1 suggests that cobalt(II) in CoCPA is five-coordinate. Similar but less extensive studies on inhibition by 2,2'-bipyridyl and 8-hydroquinoline-5-sulfonic acid have also been carried out.

Kinetics of interaction of ligands with carboxypeptidase A.

Rate constants for the interaction of a number of ligands with the active site zinc ion of carboxypeptidase A have been measured at pH 7.0, 25 degrees, 1.0 M NaCl. Polydentate ligands such as EDTA, NTA or CyDta do not accelerate the rate at which the zinc ion dissociates from the protein. Bidentate or tridentate ligands on the other hand are able to attack the zinc ion directly; the rates are first order in enzyme and first order in ligand. A mechanism for the reaction is proposed, in which a ternary complex LZnCPA is formed which rapidly dissociates into ZnL and apo CPA. Comparison of results for a variety of ligands leads to the conclusion that in the ternary complex tridentate ligands bind to the zinc ion through only two donor groups. The reaction of 1.10-phenanthroline with ZnCPA has been studied from pH 6 to 9, and a mechanism proposed which accounts for the pH profile of the reaction.

Kinetics of formation and dissociation of metallocarboxypeptidases.

The rates of formation of a number of metallocarboxypeptidases from metal ions and bovine apocarboxypeptidase A (CPA) have been measured directly and by a competitive method. Rates were determined with pH = 6-8 by utilising the pH change attending metal-ion incorporation, employing indicator and stopped-flow. Second-order rate constants Kf, M-1 s-1 at 25 degrees C, I = 1 M NaCl, pH = 7, Tris = 25 micrometer) were 1.7 X 10(5) (Mn2+), 3 X 10(4) (Co2+), 5 X 10(3) (Ni2+), 7 X 10(5) Zn2+), and 9 X 10(5) (Cd2+). Relative incorporation rate constants were determined at 25 degrees, pH = 7.0, Tris = 0.1 M, by competing two metal ions for a deficiency of apoprotein and analyzing the products by differential enzyme activity. Agreement between the two methods was reasonable. Rate constants for dissociation of CoCPA, NiCPA, and ZnCPA were measured by loss of enzyme activity on addition of the metal ion scavenger EDTA. Values of kd at 25 degrees, I = 1.0 M NaCl, pH = 7.0 were 8 X 10(-3), 3 X 10(-5), and 4 X 10(-4) s(-1), respectively. Values of K obtained kinetically (kf/kd) were in good agreement with those determined by activity measurements of equilibrated solutions. Results are compared with those of bovine apocarbonic anhydrase, where generally significantly slower rates are encountered.

Hydrolysis of substituted 8-acetoxyquinolines.

In the absence of metal ions, the hydrolysis of 2-methyl-8-acetoxyquinoline and of 5-chloro-8-acetoxyquinoline follow the same reaction paths as those of the parent ester 8-acetoxyquinoline, including an intramolecular catalysis by the quinoline nitrogen. Unlike the hydrolysis of the other esters, that of the 2-methyl compound appears not to be catalysed by metal ions, and this is consistent with the view that catalysis by a metal ion involves the formation of a 7-membered chelate structure.