Site-directed chemical cleavage

A pathway (Scheme I) (8.9) for the hydrolysis of oligoglycosides by lysozyme that differs from the previously accepted mechanism (Scheme II) (3,10-12) is described in this section. The alternative pathway, suggested by results of a 55-ps MD simulation of the lysozyme (GlcNAc)e complex (1), is consistent with the available experimental data and with stereoelectronic considerations. Experimental data have demonstrated that Glu 35 and Asp 52 are essential, as shown by recent site-directed mutagenesis results (13.) which corroborate chemical modification studies (3.14 and references cited therein), and that the reaction proceeds with retention of configuration at Ci Q and references cited therein). A fundamental feature of the alternative pathway is that an endocyclic bond is broken in the initial step, in contrast to the exocyclic bond cleavage in the accepted mechanism. 

Catalysis by (6—4) photolyase must accomplish two chemical tasks cleavage of the C6—C4 sig a bond, and transfer of the OH (or —NH2) group from the C5 of the 5 base to the G4 of the 3 base. Because formation of the (6—4) photoproduct is presumed to proceed through a four-mem-bered oxetane or azetidine intermediate, it has been proposed that (6—4) photolyase first converts the open form of the (6—4) photoproduct to the four-membered ring by a thermal reaction, and then the four-mem-bered ring is cleaved by retro [2+2] reaction photochemically (Kim et al, 1994 Zhao et aL, 1997). A site-directed mutagenesis study has identified two histidine residues in the active site that may participate in conversion of the (6-4) photoproduct to the oxetane intermediate by general acid-base catalysis (Hitomi et al, 2001). A current model for catalysis by (6-4) photolyase is as follows (Fig. 8) The enzyme binds DNA and flips out the... 

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