(575e) Determining the Limitations of Processive Polysaccharide Deconstruction By Glycoside Hydrolases

Processive glycoside hydrolases (GHs) deconstruct polysaccharides (e.g., cellulose and chitin) by associating with the substrate and repeatedly hydrolyzing glycosidic bonds without dissociating. The repetitive hydrolytic process – the processive cycle – is thought to occur in a stepwise fashion; the GH cleaves the bond, expels the dimeric product, and moves forward along the polymer chain to the next available glycosidic bond. The cycle continues until the enzyme reaches the end of a chain or a barrier and dissociates. Each step likely has an associated free energy barrier, but the entire process must be energetically favorable overall. For two processive cellulases, Hypocrea jecorina Cel7A and Cel6A, free energy calculations have previously demonstrated that the hydrolytic event is rate-limiting within the processive cycle. However, similar studies on the energetic barriers for other processive glycoside hydrolases do not exist, and it is not clear that the rate-limiting step must be the same for different GH families. As such, we do not have sufficient knowledge of processive GH reaction coordinates to provide a generalized description of processive functionality. Accordingly, we elucidated the free energy barriers along the processive cycle of a GH family 18 chitinase, Serratia marcescens ChiA, extending our knowledge to a third GH family. Molecular dynamics simulations and umbrella sampling free energy calculations were used to determine the energetic barriers of forming the ‘presliding,’ ‘sliding,’ and ‘Michaelis complex’ intermediate states in the processive cycle. Comparing these barriers to experimental kinetic rate data, we show that ChiA hydrolytic step is also rate-limiting within the S. marcescens ChiA processive cycle, temptingly suggesting this may be a general limitation of the processive GH cycle. Our findings reveal another piece of the puzzle that will enable us to purposefully manipulate enzyme function for improved enzymatic hydrolysis of biomass and oligosaccharide synthesis.