Beta-D-glucosidase reaction kinetics from isothermal titration microcalorimetry.
The cellobiase activities of nine thermal stable mutants of Thermobifida fusca BglC were assayed by isothermal titration microcalorimetry (ITC). The mutations were previously generated using random mutagenesis and identified by high-temperature screening as imparting improved thermal stability to the beta-D-glucosidase enzyme. Analysis of the substrate-saturation curves obtained by ITC for the wild-type enzyme and the nine thermally stabilized mutants revealed that the wild type and all the mutants were subject to binding of a second substrate molecule. Furthermore, the "inhibited" enzyme-substrate complexes were shown to retain catalytic activity. In the case of three of the BglC mutants (N178I, N317Y/L444F, and N317Y/L444F/A433V), binding of a second substrate molecule resulted in improved cellobiose turnover rates at lower substrate concentrations. No correlation between denaturation temperatures of the mutants and activity on cellobiose at 25 degrees C was evident. However, one particular mutant, BglC S319C, was significantly improved in both thermal tolerance and cellobiase activity with respect to those of the wild-type BglC. The triple mutant, N317Y/L444F/A433V, had a 5 degrees C increase in denaturation temperature while maintaining activity levels similar to that of the wild type at higher substrate concentrations. ITC provided a highly sensitive and nondestructive means to continuously monitor the reaction of BglC with cellobiose, resulting in abundant data sets that could be rigorously analyzed by fitting to known enzyme kinetics models. One distinct advantage of using data from the ITC was the empirical validation of the pseudo steady state assumption, a necessary condition for obtaining solutions to the proposed mechanisms.
Jeoh
Tina
T
National Renewable Energy Laboratory, National Bioenergy Center, 1617 Cole Boulevard, Golden, CO 80401, USA. tina_jeoh@nrel.gov
Baker
John O
JO
Ali
Mursheda K
MK
Himmel
Michael E
ME
Adney
William S
WS
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D002151:Calorimetry / Q000379:methods*; D002623:Chemistry Techniques, Analytical; D004795:Enzyme Stability / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D011994:Recombinant Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D001617:beta-Glucosidase