Determination of substrate binding energies in individual subsites of a family 18 chitinase

Abstract

a b s t r a c tThermodynamic parameters for binding of N-acetylglucosamine (GlcNAc) oligomers to a family 18 chitinase, ChiB of Serratia marcescens, have been determined using isothermal titration calorimetry. Binding studies with oligomers of different lengths showed that binding to subsites À2 and +1 is driven by a favorable enthalpy change, while binding to the two other most important subsites, +2 and +3, is driven by entropy with unfavorable enthalpy. These remarkable unfavorable enthalpy changes are most likely due to favorable enzyme-substrate interactions being offset by unfavorable enthalpic effects of the conformational changes that accompany substrate-binding.

Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation

“…In a future study, we will examine the role of aromatic residues in corresponding processive and nonprocessive enzymes via a similar TI approach. Moreover, experimental measurements using techniques such as isothermal titration calorimetry (22,76,77), could potentially validate the trends observed in the changes in ligand binding free energy calculated here with TI. However, this would require substrates with modified linkages (e.g.…”

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

“…2A). In addition to the steric constraints of the tunnel, carbohydrate-interactions of aromatic residues lining the substrate-binding clefts are important for processivity and, more generally, may make large contributions to the ligand binding free energy (21,22,25,26,28,(62)(63)(64)(65). Notably, ChiC2 has fewer such aromatic residues than ChiA and ChiB, primarily affecting the product subsites (Fig.…”

Thermodynamic Relationships with Processivity in Serratia marcescens Family 18 Chitinases

“…His 10 -ChiB-E144Q is previously constructed. 38 To subclone ChiA-E315Q 37 and ChiC-E141Q into the vector pET16b (Novagen, Madison, WI, U.S.A.), the Chitinase fragments were amplified by PCR using primers listed in Proteins were purified on a column packed with Ni-NTA Agarose matrix (Qiagen, Venlo, Netherlands; 1.5 cm in diameter, 5 mL stationary phase in total). The column was pre-equilibrated in a buffer containing 20 mM Tris-HCl, 20 mM imidazole, and 500 mM NaCl at pH 8.0 before the periplasmic and cytoplasmic extracts were applied.…”

“…ChiA-E315Q and ChiB-E144Q are previously constructed. 37,38 ChiC-E141Q was prepared using the QuikChange site directed mutagenesis kit from Stratagene (La Jolla, CA, U.S.A.), as described by the manufacturer. The primers used for the mutagenesis are listed in Table 1 and were purchased from Life Technologies (Carlsbad, CA, U.S.A.).…”