Enzymatic activity of Trp 62-modified lysozyme

The time-courses of action of Trp 62-modified lysozymes on the initial substrate chitopentaose were measured by means of high-performance gel-filtration. The activities of the modified lysozymes, represented by the rate of disappearance of the initial substrate (overall rate) were lowered to various extents depending on the method of the modification. On the other hand, the time-courses were calculated by changing the values of rate constants, using the binding free energy of each subsite estimated by the optimization technique (Kuhara et al. (1982) J. Biochem.). For NBS- and NPS-lysozymes, the calculated time-courses were not in good agreement with the experimental ones, when the binding free energies estimated by the optimization technique were used for the calculation. Therefore, the binding free energies of the subsites were estimated from the experimental time-courses with the assumption that the values of the rate constants do not change upon modification of Trp 62 in subsite C. As a result, it was found that, though the modification was at subsite C, the binding free energy of subsite A was profoundly lowered, while that of subsite B remained almost unchanged.