ABSTRACT

Within a few years of its discovery CPD A was demonstrated to bind a second zinc in the pH range 7-10 by equilibrium dialysis [160].

Kinetic studies show that zinc and lead both competitively inhibit peptidase catalysis in the micromolar range [161]. The apparent inhibition constant for zinc is 5.2 pM at pH 8 . This inhibition is strongly pH-dependent (Fig. 8). Over the pH range 510 zinc is distributed among five protonation states, Zn2+ cation, Zn-mono-, di-, tri-, and tetrahydroxide. The inhibition pattern follows closely the formation curve for the zinc monohydroxide species for the pH range 6.5-8. In this pH region the active form of the enzyme, EH, predominates. Above 9 inhibition decreases as the zinc monohydr­ oxide species is converted to its higher di-, tri-, and tetrahydroxide forms. In the pH region below 6.5, the strength of the zinc inhibition is suppressed below the level

expected solely for the formation of the Zn monohydroxide species. However, this is the pH region in which the active enzyme form EH is converted to EH2 [162]. The experi­ mental data fits very nicely to a mechanistic scheme where inhibition occurs through the binding of the zinc monohydroxide species to the active EH species of the enzyme (Fig. 9). The pH-independent constant for this zinc inhibition is 0.71 pM. The derived pKa of 6 for the inhibition studies agrees with the corresponding value obtained in peptide hydrolysis experiments for the group, EH2, whose ionization leads to forma­ tion of the eatalytically active form of the enzyme [162].