STRUCTURAL-ANALYSIS OF MUTANT HEN EGG-WHITE LYSOZYME PREFERRING A MINOR BINDING MODE

Trp62 in hen egg-white lysozyme has general features observed in prote in-carbohydrate interactions, a stacking interaction toward nonpolar s urface of the substrate sugar residue B and a hydrogen bonding network with the residue C, Our previous report (I. Kumagai, K. Maenaka, F. S unada, S. Takeda, K. Miura, Eur. J. Biochem. 212 (1993) 151-156.) show ed that the substitution of Trp62 changed the substrate binding modes, especially, the Trp62His mutant exhibited the drastic change of the b inding mode and preferred to a minor binding mode of the wild-type enz yme. In order to clarify the relationship between functional and struc tural changes of the Trp62His mutant, we analyzed the structure of the Trp62His mutant hen lysozyme complexed with the substrate analogue, ( GlcNAc)(3), by X-ray crystallography. The overall protein structure in the mutant lysozyme complex was almost identical to that in the wild- type. His62 shared almost the same plane as the indole ring of Trp62. of the wild-type. Although the (GlcNAc)(3) molecule which is an inhibi tor against the wild-type lysozyme was cocrystallized, the Trp62His mu tant did not put it in the sites A-B-C but hydrolyzed it as a substrat e. One of the products, (GlcNAc)(3), whose reducing end is alpha-anome r, was bound in another binding mode sticking out from the active-site cleft. The hydrolytic activity against the synthetic substrate showed that the mutant was a beta-anomer retaining enzyme, so the alpha-anom er product was converted from the beta-anomer product. Therefore, the Trp62His mutant showed the remarkable change of the substrate binding modes not by alteration of the catalytic system but possibly by subtle rearrangement of general features of protein-carbohydrate interaction s between His63 and the sugar residues B and C. (C) 1998 Elsevier Scie nce B.V.