The inhibitory effect of various substances on an immobilized derivative of carboxypeptidase CN (AEC-CPase CN), which was prepared by conjugating the enzyme to AE-cellulose through glutaraldehyde, was examined. Like the native enzyme, AEC-CPase CN was inactivated by methanol and this inactivation became more pronounced as the concentration of methanol increased. AEC-CPase CN was slightly more stable to the attack of Pronase than the native enzyme. Enzymic activity of AEC-CPase CN was completely loss by the addition of low concentrations of sodium dodecyl sulfate and cetyltrimethylammonium bromide, whereas Triton X-100 and Tween 80 caused only a partial loss of the enzymic activity even at high concentrations. Of six phenylalanine analogs tested, β-phenylpropionic acid, a competitive inhibitor, showed the most inhibitory effect. Pretreatment of AEC-CPase CN with 25 mM β-phenylpropionic acid almost completely protected the enzyme from inactivation by diisopropylfluorophosphate, while the attack of p-bromophenacyl bromide and HgCl2. was only partially blocked by the same pretreatment. The denaturating effect of methanol, heat, and pH was not prevented by the pretreatment with β-phenylpropionic acid.
