Tuesday, May 3, 2011
The processive Serratia marcescens chitinases A (ChiA) and B (ChiB) are thought to degrade chitin in the opposite directions. Mutation studies with ChiB have suggested that processivity is governed by aromatic residues in the +1 and +2 (aglycon) subsites close to the catalytic center [1]. Mutational studies of ChiA have shown that processivity is primarily governed by Trp167 which is located in the -3 (aglycon) subsite and which is absent in ChiB [2]. Thus, aromatic residues located in subsites close to the catalytic centre and on the side where the polymeric part of the substrate is thought to bind during processive action are crucial for maintaining processivity. Remarkably, whereas non-processive mutants of ChiA and ChiB consistently showed reduced efficiency towards crystalline chitin, they showed up to 20-fold higher activities toward chitosan, a soluble polymeric chitin-derivative. These results show that the processive mechanism is essential for an efficient conversion of crystalline substrates but comes at a large cost in terms of intrinsic enzyme speed. Recently, we have carried out several studies on the kinetic and binding properties of ChiA and ChiB, providing further insight into directionality-related differences in active site architecture and into the links between processivity and enzyme speed.
[1] Horn, S.J., Sikorski, P, Cederkvist, J.B., Vaaje-Kolstad, G., Sørlie, M., Synstad, B., Vriend, G., Vårum, K.M., Eijsink, V.G.H., 2006, PNAS, 103 (48), 18089-18094.
[2] Zakariassen, H., Aam, B.B., Horn, S.J., Vårum, K.M., Sørlie, M., Eijsink, V.G.H., 2009,. Journal of Biological Chemistry, 284, 10610-10617.