Carboxypeptidase A: Approaches to the Chemical Nature of the Active Center and the Mechanisms of Action

in 0.02 M sodium Veroral-2 M NaCI, pH 7.5 buffer at 0? for a reaction time of thirty min. The pH of the reaction was maintained at 7.5 by the addition of 1 M NaOH by means of a pH Stat. Excess anhydride was removed by dialysis against the same buffer at 0? for 48 hr. [odinations were performed with a 35-fold molar excess of 12 in 0.5 M KI at 25? for ten min. Photooxidations were carried out in the presence of methylene blue according to the method of Weil and Buchert.7 All chemicals used in these experiments were reagent grade and used without further purification except for acetic anhydride which was redistilled. Results and Discussion.-The metal binding site of apocarboxypeptidase: Ag+, p-mercuribenzoate, or ferricyanide titrate only one -SH group in the apocarboxypeptidase and none in the metallocarboxypeptidasel2 (Table 1). A peptide containing the single cysteine residue of carboxypeptidase to which zinc is bound has recently been isolated. The sequence and magnitude of the stability constants of different metallocarboxypeptidases, their comparison to those of simple ligands and complexometric titrations indicated early that the metal atom is also bound to nitrogen.' 12 The experiments suggesting that the a-amino group of the N-terminal asparagine (AspNH2) (Table 1) may be the second donor group for zinc binding are quite analogous to those employed for the -SH group. On exposure to fluorodinitrobenzene or phenylisothiocyanate, the apoenzymel readily forms DNP-aspartic acid (DNP-Asp) or PTH-asparagine (PTN-AspNH2), respectively, while the yields from the metalloenzyme are significantly less. Zinc appears to be displaced from the enzyme in proportion to the reaction of these reagents with the N-terminal asparagine. The sum of the number of gram atoms of zinc bound per mole of