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Endoglucanase A from Cellulomonas fimi : determination of the amino acids directly involved in catalysis and their function

University of British Columbia

The overall objective of this study was to characterize the catalytic domain of endoglucanase A (CenA) from Cellulomonasfimi. More specifically, the objective was to identify the amino acids in the catalytic domain which are directly involved in acid and base catalysis and/or in stabilizing the transition state. The catalytic domains of 13-1,4-glucanases can be grouped into families of related amino acid sequences. CenA is a member of family 6. All enzymes from this family are believed to hydrolyze 13-1,4-glucosidic bonds using a general acid-base catalytic mechanism resulting in inversion of anomeric configuration at the scissile bond. Three-dimensional structures for two cellulases from family 6 have been determined by x-ray crystallographic analysis. These structures show that there are four aspartate residues which are in a position to function as acid catalyst, base catalyst and/or transition state stabilizers. These aspartates are conserved in all members of family 6. The roles of D216, D252, D287 and D392, the corresponding amino acid residues in CenA, were determined. These aspartates have been systematically replaced with alanine and glutamate via site-directed mutagenesis and the resulting effects on activity, pH profile, substrate specificity and overall structure have been determined. Changes in overall structure were monitored using circular dichroism spectroscopy and no significant differences between the wild-type and mutant proteins were found. Active site structure was also found to be intact as all proteins bound to a cellobiose affinity column. The kinetic parameters of the enzymes were determined on various substrates with different leaving groups. Further kinetic analysis of mutants using small nucleophilic anions and using a- and f3-cellobiosyl fluoride, as well as pH dependence studies, were also carried out. On the basis of these results, D252 and D392 are assigned as the acid and base catalysts, respectively, in CenA. Residue D287 appears to aid D252 in acid catalysis and D216 is not absolutely required for catalysis.

Thesis/Dissertation

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2009-06-04

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http://hdl.handle.net/2429/8789

Microbiology and Immunology

University of British Columbia

UBCV

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