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Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories
Author(s) -
Taylor Marc G.,
Kirsch Jack F.,
Rajpal Arvind
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070902
Subject(s) - lysozyme , chemistry , crystallography , metastability , docking (animal) , reaction rate constant , stereochemistry , kinetic energy , kinetics , physics , biochemistry , medicine , nursing , organic chemistry , quantum mechanics
The rate constants, k on, for the formation of hen (chicken) lysozyme (HEWL) Fab‐ l0 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 10‐fold range [(0.18 to 1.92) × 10 6 M −1 s −1 ]. The affinity constants, K D , cover a broader, 440‐fold, range from 0.075 to 33 nM. Values of K D as high as 7 μM 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 on are negatively correlated with side‐chain volume at position 101 HEWL , but are essentially independent of this parameter for position 21 hewl substitutions. The multiple mutations made at positions 21 HEWL and 101 HEWL provide sufficient experimental data on complex formation to evaluate Φ values [Φ = (ΔΔG ‡ on )/(ΔΔG D )] 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 2l HEWL (Φ avg = 0.2) is encounter complex formation, but the larger Φ avg value of 0.36 experienced for most position 10l HEWL mutations indicates a larger contribution from the post‐encounter annealing process at this site for these replacements.