COMPARISON OF THE STRUCTURES OF 3 CARBOXYPEPTIDASE-A PHOSPHONATE COMPLEXES DETERMINED BY X-RAY CRYSTALLOGRAPHY

The structures of the complexes of carboxypeptidase A (CPA) with two tight-binding phosphonate inhibitors have been determined by X-ray crystallography. The inhibitors, Cbz-Phe-Val(P)-(O)-Phe[ZFV(P)(O)F] and Cbz-Ala-Gly(P)-(O)-Phe[ZAG(P)(O)F], bind noncovalently to CPA with dissociation constants (K(i)'s) of 11 fM and 710 pM, respectively. The CPA-ZFV(P)(O)F complex crystallizes in the space group P2(1)2(1)2(1) with unit cell parameters a = 65.3 angstrom, b = 63.4 angstrom, and c = 76.0 angstrom, and the CPA-ZAG(P)(O)F complex crystallizes in the space group P2(1)2(1)2(1) with unit cell parameters a = 63.4 angstrom, b = 65.9 angstrom, and c = 74.4 angstrom. Both structures were determined by molecular replacement to a resolution of 2.0 angstrom. The final crystallographic residuals are 0.189 for the CPA-ZFV(P)(O)F complex and 0.191 for the CPA-ZAG(P)(O)F complex. The CPA-ZFV(P)(O)F complex exhibits the lowest K(i) yet determined for an enzyme-inhibitor interaction. Comparison of the CPA-ZFV(P)(O)F structure with that of the CPA-ZAA(P)(O)F complex [Kim, H., & Lipscomb, W. N. (1990) Biochemistry 29, 5546-5555] indicates the likely important contributions of hydrophobic and weakly polar enzyme-inhibitor interactions to the exceptional stability of the CPA-ZFV(P)(O)F complex. Among these interactions is a network of four aromatic rings of CPA and ZFV(P)(O)F in a configuration that allows stabilizing aromatic-aromatic edge-to-face interactions from one ring to the next. A comparison of the structures of the CPA-ZFV(P)(O)F, CPA-ZAA(P)(O)F and CPA-ZAG(P)(O)F complexes shows that all three phosphonates assume a similar binding mode in the active-site binding groove of CPA. For ZAG(P)(O)F, the glycyl P1 residue does not lead to an anomalous or a partially disordered binding mode as seen in some previous complexes of CPA involving dipeptide analogue inhibitors with glycyl P1 residues. The additional enzyme-inhibitor interactions for these tripeptide phosphonates secure a binding mode in which a P(i) portion of the inhibitor is clearly bound by the corresponding S(i) binding subsite. These three phosphonates have been implicated as transition-state analogues of the CPA-catalyzed reaction. The phosphinyl groups of these phosphonates coordinate to the active-site zinc in a manner that has been proposed as a characteristic feature of the general-base (Zn-hydroxyl or Zn-water) mechanism for the CPA-catalyzed reaction. Further mechanistic proposals are made for Arg-127, whose probable role in binding substrates is apparent in these CPA-phosphonate complexes.