Simple and reliable immobilization techniques that preserve the activity of enzymes are of interest in many technologies based on catalysis. Here, two redox enzymes, glucose oxidase from Aspergillus niger and horseradish peroxidase, were immobilized by physisorption on glassy carbon electrodes coated with Schizophyllum commune hydrophobin. Hydrophobins are small, interfacially active proteins that have the remarkable property of adhering to almost any surface. We showed recently that these proteins can be used to immobilize small, electroactive molecules. The results obtained in this work show a way to easily manufacture stable, enzyme-based catalytic surfaces for applications in biosensing.
Subspecies defining the maturation pathway of bovine chymotrypsinogen to alpha-chymotrypsin have been separated in a single chromatographic run by affinity to iminodiacetic acid-Cu(II) [IDA-Cu(II)] immobilized onto Novarose. A major highlight of the elution pattern is that, as maturation proceeds, these subspecies exhibit a correlated increase in affinity toward IDA-Cu(II). This behavior is analyzed by a combination of physicochemical and molecular modeling techniques to assess the contribution of the two histidines present in chymotrypsins, at positions 40 and 57 on the protein surface. Catalytic His-57 features adequate surface accessibility to serve as a ligand to IDA-Cu(II), but its participation is clearly ruled out by specific chemical modification. In contrast, His-40, whose side chain is buried in the crystal structures of both zymogen and mature enzyme, surprisingly proves the most plausible candidate as an electron donor to IDA-Cu(II). This apparent conflict between histidine accessibility and their implication in IDA-Cu(II) recognition has been rationalized on the basis of their flexibility and/or hydrogen-bonding status, with the following outcome. First, histidine constitutes a useful reporter group for subtle protein conformational fluctuations. Second, static accessibility computation alone provides no unequivocal guideline as to whether a protein residue can serve as a ligand. Third, this study is the first to document the occurrence of a screening effect due to hydrogen bonding of an otherwise "accessible" histidine. A significant corollary to this finding would be that the catalytic histidine is rigidly entrapped in a remarkably strong hydrogen-bonding network, a situation that may pertain to mechanistic aspects of catalysis.
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