In an effort to expand the catalytic repertoire of antibodies to encompass metal ion-assisted reactions, three classes of metal ion-binding sites were designed within the antigen-binding site of catalytic single-chain antibody (SCA) 43C9 and characterized by a variety of spectroscopic techniques. With structural motifs of metalloenzymes as prototypes, computer modeling techniques were used to design these sites. The affinities of each class of metal ion-binding sites for a variety of divalent metal ions were determined by fluorescence quenching techniques. One class of binding sites, consisting of His residues at positions L32, L34, and L91, bound Zn(II) with a KDvalue of 3.3 ± 0.8 μM; however, the affinity for the inducing antigen was decreased by at least 104 relative to that of the wild-type in the absence of Zn(II). The second class of metal ion-binding sites, which consisted of His residues at positions H33, H35, and H95, possessed greater than 100-fold selectivity for Zn(II) over any other divalent metal ion tested and bound this ion with KDvalues of 1.5—3.7 μM. The third class of metal ion-binding sites utilized His residues at positions L91 and L96 and, in some cases, H95. This class was selective for Cu(II) over Zn(II), binding the former with KD values of 0.5—2.1 μM and the latter with KDvalues of 10–40 μM. Continuous-wave EPR studies of Cu(II) bound to this class of mutants verified the results of the fluorescence quenching assays; Cu(II) binding resulted in EPR signals that were well approximated by a simulation using the parameters of A||= 166.0G, A⊥ = 6.0G, g||= 2.19, and g⊥= 2.05. Furthermore, pulsed EPR experiments (ESEEM) demonstrated that (1) a low-affinity Cu(II) site (KD≥100 μM) consisting of a single His residue existed in the wild-type SCA, (2) two His residues acted as Cu(II) ligands in the R-L96-H single mutant, and (3) three His residues acted as Cu(II) ligands in the R-L96-H, Y-H95-H double mutant. These results are consistent with the original computational design. In addition, UV—vis studies suggested a rare Tyr to Cu(II), ligand-to-metal charge transfer band at 490 nm (∊ = 55 M-1 cm-1) that arose from coordination of the Tyr H95 side chain to the bound Cu(II). As expected, this band was absent in the Cu(II)-bound form of the R-L96-H, Y-H95-F double mutant SCA. Finally, the R-L96-H mutant was shown to simultaneously bind metal and p-nitrophenol.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry