Carboxypeptidase CN. II. Stability and some chemical and kinetic properties.

Abstract

Carboxypeptidase CN [EC 3.4.12.1] is composed of 703 amino acid residues, 32 hexose residues (as galactose), and 5 hexosamine residues (as glucosamine): these are Trp6,Lys29, His18, Arg44, Asp83, Thr37, Ser52, Glu46, Pro14, Gly43, Ala50, Cys(half)6, Val44, Met7, Ile58, Leu93, Tyr37, Phe36, Gal32, and GlcN5 The enzyme was stable in the pH range from 5.2 to 5.7 and in the temperature range from 0 to 45°C. At pH values below 4.0 or over 7.0, the enzyme was quickly denatured. Its activity considerably decreased on lyophilization or desalting. It was completely inhibited by the detergents SDS and CTMA, but not by Brij-35 or Tween 80. The enzyme was also inactivated by urea, GlyOMe (in the presence of EDC), p-BPB, phenylglyoxal, and PMSF. The rate of hydrolysis of Z-Glu-Phe by the enzyme increased with increasing concentration of the substrate up to 2.7 ×10−2M, but in the concentration range from 3.6 ×10−2 to 4.4 ×10−2M, it decreased with increasing concentration of the substrate.The values of Km and Vmax for the hydrolysis of Z-Glu-Phe were 4.0 ×10−2M and 2.3 ×10−4M, mole/liter-min-mg, respectively. Inactivation of the enzyme with DFP and HgCl2 resulted in a decrease in the value of Vmax, whereas that of Km remained unchanged, indicating that DFP and HgCl2 are both noncompetitive inhibitors. β-Phenylpropionic acid, on the other hand, inhibited the enzyme reversibly and increased the value of Km without changing that of Vmax significantly, showing that the compound is a competitive inhibitor.