Phosphonate Analogs of Carboxypeptidase A Substrates are Potent Transition-State Analogue Inhibitors

Analogues of tri- and tetrapeptide substrates of carboxypeptidase A in which the scissile peptide linkage is replaced with a phosphonate moiety (-PO2--O-) were synthesized and evaluated as inhibitors of the enzyme. The inhibitors terminated with either L-lactate or L-phenyllactate [designated (O)Ala and (O)Phe, respectively] in the P1’ position. Transition-state analogy was shown for a series of 14 tri- and tetrapeptide derivatives containing the structure RCO-AlaP-(O)Ala [RCO-AP(O)A, AP indicates the phosphonic acid analogue of alanine] by the correlation of the Ki values for the inhibitors and the Km/kcat values for the corresponding amide substrates. This correlation supports a transition state for the enzymatic reaction that resembles the tetrahedral intermediate formed upon addition of water to the scissile carbonyl group. The inhibitors containing (O)Phe at the P1’ position proved to be the most potent reversible inhibitors of carboxypeptidase A reported to date: the dissociation constants of ZAFP(O)F, ZAAP(O)F, and ZFAP(O)F are 4, 3, and 1 pM, respectively. Because of the high affinity of these inhibitors, their dissociation constants could not be determined by steady-state methods. Instead, the course of the association and dissociation processes was monitored for each inhibitor as its equilibrium with the enzyme was established in both the forward and reverse directions. A phosphonamidate analogue, ZAAPF, in which the peptide linkage is replaced with a –PO2--NH- moiety, was prepared and shown to hydrolyze rapidly at neutral pH (t1/2 = 20 min at pH 7.5). This inhibitor is bound an order of magnitude less tightly than the corresponding phosphonate, ZAAP(O)F, a result that contrasts with the 840-fold higher affinity of phosphonamidates for thermolysin [Bartlett, P. A,, & Marlowe, C. K. (1987) Science 235, 569-5711, a zinc peptidase with a similar arrangement of active-site catalytic residues