KIRSCH complex : Delineation of docking trajectories

The rate constants, k,,, for the formation of hen (chicken) lysozyme (HEWL) .Fab-lO complexes have been determined for wild-type (WT) and epitope-mutated lysozymes by a homogeneous solution method based on the 95% reduced enzymatic activity of the complex. The values fall within a narrow IO-fold range [(0.18 to 1.92) X lo6 M" s I 1. The affinity constants, KO. cover a broader, 440-fold, range from 0.075 to 33 nM. Values of KO as high as 7 p M were obtained for the complexes prepared from some mutations at HEWL positions 96 and 97, but the associated kinetic constants could not be determined. The values of k,, are negatively correlated with side-chain volume at position IOIHEWL, but are essentially independent of this parameter for position 21HEWL substitutions. The multiple mutations made at positions 21HEWL and ~ O ~ H E W L provide sufficient experimental data on complex formation to evaluate @ values [@ = (AAGi;,)/(AAG,)] at these two positions to begin to define trajectories for protein-protein association. The data, when interpreted within the concept of a two-step association sequence embracing a metastable encounter complex intermediate, argue that the rate determining step at position 2lHEWL (aavg = 0.2) is encounter complex formation, but the larger @'a\.g value of 0.36 experienced for most position 10l~EwL mutations indicates a larger contribution from the post-encounter annealing process at this site for these replacements.