Related Experiment Video
Updated: Jun 5, 2026

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases
Published on: December 23, 2022
Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger
Sven Cuyvers1, Emmie Dornez, Mohammad N Rezaei
1Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Leuven, Belgium.
Abstract:
The secondary substrate binding site (SBS) of Bacillus subtilis and Aspergillus niger glycoside hydrolase family 11 xylanases was studied by site-directed mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates. Modification of the SBS resulted in an up to three-fold decrease in the relative activity of the enzymes on polymeric versus oligomeric substrates and highlighted the importance of several amino acids in the SBS forming hydrogen bonds or hydrophobic stacking interactions with substrates. Weakening of the SBS increased K(d) values by up to 70-fold in binding affinity tests using natural substrates. The impact that modifications in the SBS have both on activity and on binding affinity towards polymeric substrates clearly shows that such structural elements can increase the efficiency of these single domain enzymes on their natural substrates.
Related Concept Videos
Enzymes
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Introduction to Mechanisms of Enzyme Catalysis
Ligand Binding and Linkage
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Enzyme Kinetics
Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is...
Enzyme Inhibition

