Reprin\.ed from COLD SPRING HARllOR SYMPOSIA ON QUANTITATIVE mOLOGY

One of the most fascinating features of the mechanism proposed by Phillips and co-workers (Blake et aI., 1967a, b; Phillips, 1966) for the cleavage of oligosaccharides by lysozyme is the distortion of the saccharide bound in subsite D into a half-chair conformation. This mechanism is based upon X-ray crystallographic studies of the non-productive complex of lysozyme with chitotriose, GlcNAc-,B(l -+ 4)-GlcNAc-,B(1 -+ 4)GlcNAc, (bound in subsites A, B, C) and model building. The involvement of substrate distortion in the proposed mechanism is supported by several studies (Chipman et aI., 1967; Chipman and Sharon, 1969; Chipman, 1971; Johnson et aI. , 1968; Rupley and Gates, 1967) which estimate that the contribution to the free energy of binding of oligosaccharides from the saccharide bound in subsite D is unfavorable by 3-6 kcal mole-I. This is exemplified by a comparison of the binding constants for GlcNAc-,B(1 -+ 4)-MurNAc-,B(1 -+ 4)GlcNAc (KA = 2.8 X 105 M-I) and GlcNAc-,B(1 -+ 4)MurNAc-,B(I-+ 4)-GlcNAc-,B(I-+ 4)-MurNAc (KA = 2.1 X 103 ~I) (Chipman and Sharon, 1967). With an estimate of the favorable interactions at subsite D, the energy available for distortion is 6-12 kcal mole-I (Johnson et aI., 1968) , which is approximately that required for the distortion of cyclohexanes from the chair to the half-chair conformation (Eliel et aI., 1965). Since the hydrolysis of natural oligosaccharides by lysozyme is complicated by non-productive binding and tmns-glycosylation, the study of any one feature of the mechanism is difficult without the use of synthetic substrates (Rand-Meir et aI., 1969). In agreement with the mechanism proposed by Phillips and co-workers, Dahlquist et al. (1968, 1969) have concluded that considerable carbonium ion character is involved in the hydrolysis of the