STUDIES ON THE ENZYMIC ACTIVITY OF LYSOZYME, II. THE HYDROLYSIS AND TRANSFER REACTIONS OF N-ACETYLGLUCOSAMINE OLIGOSACCHARIDES*.

The succeeding paper of this series' considers the chemistry of the binding of substrate to lysozyme, and discussed here are certain details of the cleavage of the oligosaccharides of N-acetylglucosamine. Hydrolysis by lysozyme of this type of substrate was first observed by Berger and Weiser2 for chitin, the high-molecular-weight 3-(1-4)-linlked polymer of N-acetylglucosamine, and subsequent reports have demonstrated cleavage of the trimer,3 4 tetramer,5 and pentamer.4 Glycosyl transfer has recently been shown to obtain with oligosaccharides derived from both N-acetylglucosamine6 and from bacterial cell walls.7 Although it has not been possible to approach the cleavage process directly using X-ray analysis, Phillips, North, Blake, and their co-workers8 have proposed a mechanism of action for the enzyme which is based upon the crystallographic structure of the tri-(N-acetylglucosamine)-lysozyme complex; a summary of their argument follows. Since the trisaccharide complex is stable and its two glycosidic bonds do not lie near enzyme groups which might plausibly be catalysts of the hydrolysis, it is likely the trimer is bound in a fashion which cannot lead to cleavage. However, an outstanding feature of the molecule is a cleft which is shown by the crystallography to be only half-filled by the trisaccharide, suggesting the filling of the remaiinder of the c-left with substrate. A total of three more units (six in all) canl be fitted inl a satisfying manner (Fig. 1). Inl the hexasaccharide complex the C1-O bond between the fourth and fifth units from the Ilolireducilig en(l (betweenI uniits D and 1E) is embraced by two carboxyl groups. Glutamic 35 forms a hydrogen bond with the bridge oxygen, and a carboxyl oxygen of aspartic 52 makes contact with the 1-carbon aIld the ring oxygen of unit, D. Cleavage of the D-E bond might therefore proceed through: (1) protoniatioin of the bridge oxygen; (2) rupture of the C1-O bond to form a C1-carbonium iOIl with the rate of reaction being enhanced by the neighboring negative charge of aspartic 52; (3) dissociation of the aglycone and its replacement by either water or a saccharide acceptor, followed by reversal of the preceding steps. In the crystallographic model, all the saccharide units except D could be placed in the cleft in chair conformations, but unit D required distortion so that the 6-carbon approached anl axial position, resulting in a high-energy conformation resembling the half-chair. Owing to distribution of the positive charge betweeii the 1-carboll and the ring oxygen, the proposed carboniium-ion inltermediate would have a similar structure,9 and the forcing of the substrate into this structure prior to development of the carbonium ion should make a major contribution to the catalysis. The foregoing mechanism based upon the crystallography is capable of explaining the kinetic data reported inl this paper, and two (correlations are of particular interest: (1) the enizyme-substrate complexes inl which cleavage can occur are much weaker than the trisaccharide-enizyme complex, inl a(c(cord with the proposed (listortioni of the rinig of uinit1 D; (2) both the chain-lenigth dependence of the reaction rate and the preferential attack by the enzyme on (certain bonds in polymeric substrates are explained by cleavage between units D and E of a hexasaccharide complex.