High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis

Chitinase A (ChiA) from the bacterium Serratia marcescens is a hydrolytic e nzyme, which cleaves beta -1,4-glycosidic bonds of the natural biopolymer c hitin to generate di-N-acetyl-chitobiose. The refined structure of ChiA at 1.55 Angstrom shows that residue Asp313, which is located near the catalyti c proton donor residue Glu315, is found in two alternative conformations of equal occupancy. In addition, the structures of the cocrystallized mutant proteins D313A, E315Q, Y390F, and D391A with octa- or hexa- N-acetyl-glucos amine have been refined at high resolution and the interactions with the su bstrate have been characterized. The obtained results clearly show that the active site is a semiclosed tunnel. Upon binding, the enzyme bends and rot ates the substrate in the vicinity of the scissile bond. Furthermore, the e nzyme imposes a critical "chair" to "boat" conformational change on the sug ar residue bound to the - 1 subsite. According to our results, we suggest t hat residues Asp313 and Tyr390 along with Glu315 play a central role in the catalysis. We propose that after the protonation of the substrate glycosid ic bond, Asp313 that interacts with Asp311 flips to its alternative positio n where it interacts with Glu315 thus forcing the substrate acetamido group of - 1 sugar to rotate around the C2-N2 bond. As a result of these structu ral changes, the water molecule that is hydrogen-bonded to Tyr390 and the N H of the acetamido group is displaced to a position that allows the complet ion of hydrolysis. The presented results suggest a mechanism for ChiA that modifies the earlier proposed "substrate assisted" catalysis.