To determine the specific roles and mechanism of action of both cellobiohydrolases (CBH I and CBH II) of Trichoderma reesei on crystalline and soluble substrates. We expect to define the role of the key amino acids in their active sites, to map the structural determinants that provide distinctive cellobiohydrolase or endoglucanase activities, and to determine the soluble and insoluble end-products produced from crystalline cellulose by CBH I and CBH II.
CBH II: The enzymatic characterization of CBHII mutant Y169F suggested that conserved tyrosine 169 may promote ring distortion at the -1 binding site. The competitive inhibitors Glc-beta-(1,4)-Xyl-1beta-(meta-iodo) benzyl and 1beta-(meta-iodo) benzylcellobioside were synthesized to test this hypothesis. Direct crystallographic evidence for ring distortion at this binding site has been obtained by structure determinations of a number of complexes at high resolution (2.0Å and better). Mutagenesis of a tryptophan, W272, located on the catalytic domain outside the tunnel entrance revealed that this residue forms the sixth glucose binding site and is required for the action of CBH II on crystalline but not on soluble substrates. An HPLC analysis of the soluble products released from polymeric substrates by wild type CBH II revealed that cellobiose is the main soluble product and suggested that CBH II makes on average 5-10 cuts before releasing a cellulose chain.
CBH I: Mutagenesis and enzymatic analysis of the proposed active site acidic residues confirmed the assignment that E212 is the nucleophile and E217 is the general acid/base in the catalytic mechanism. The deglycosylated proteins were crystallized and their structures determined at high resolution (approximately 1.7Å), and in complex with various cello-oligosaccharides. This allowed us to construct a model for a cellulose chain threading through the active site tunnel (from sites -7 to +4). The model confirms the directionality of the cellulose chain and that the complete tunnel can be used for cellulose binding. Wild type CBH I was affinity labelled using w-epoxybutyl-beta-cellobioside. Comparative liquid chromatography coupled on-line to electrospray ionization mass spectrometry were used to identify the labelled peptide, and the identity of the modified amino acid residue was determined. The specific and irreversible inactivation of CBH I was achieved by covalent modification of E212, the putative nucleophile in the hydrolysis mechanism. To determine the importance of one of the active site loops of CBH I, four different mutations were designed and the mutant proteins expressed and partially purified from an engineered T. reesei strain.
A number of new substrates were prepared for CBH kinetic studies: never-dried microcrystalline bacterial cellulose of a DPn similar to Avicel, cello-oligosaccharides of a DP up to cello-octaose, and water insoluble cellulose fragments with narrow molecular weight distribution (+/- 3 glucose units) covering the DPn-range between 9 and 16 beta-1,4 linked glucose units.
MAJOR SCIENTIFIC BREAKTHROUGHS:
- Unequivocal evidence for glucose ring distortion in the -1 site of CBH II.
- Identification of an essential determinant for crystalline cellulose degradation by CBH II.
- Identification of the catalytic residues of CBH I.
- A detailed atomic model describing how a cellulose chain binds to the active site tunnel of CBH I.