DISULFIDE ARRANGEMENT AND FUNCTIONAL DOMAINS OF BETA-1,4-ENDOGLUCANASE-E5 FROM THERMOMONOSPORA-FUSCA

Thermomonospora fusca cellulase E5 contains six cysteine residues. The number and location of the disulfide bonds and the effect of reductio n of the disulfides and modification of the resulting half-cystine res idues on enzymatic activity were determined. No free sulfhydryl groups were found in E5. Reduction and subsequent labeling with iodoacetamid e of E5 and of an enzymatically active 32-kDa proteolytic derivative o f E5 (E5cd) showed that one of the three disulfides is accessible to r eduction under nondenatured conditions while the other two are not acc essible. Full reduction of the disulfides and complete carboxymethylat ion of the six cysteines decrease the specific activity of E5 on CMC b y more than half, but reduction of only the exposed disulfide bond doe s not affect enzymatic activity or binding of E5 to cellulose. A 14-kD a proteolytic fragment of E5 containing 120 amino acids from the N-ter minus of the protein was shown to bind to crystalline cellulose. This confirms earlier evidence that the cellulose binding domain of E5 is l ocated at the N-terminus of the protein. This 14-kDa fragment contains the accessible disulfide bond involving Cys93 and Cys100. The locatio n of the two disulfide bonds in the other fragment (E5cd) was determin ed by cleaving it with cyanogen bromide under conditions that left the disulfide bonds intact. The resulting peptides were separated under b oth nonreducing and reducing conditions using RP-HPLC. Amino acid anal ysis of peptide peaks indicated that one disulfide linkage in E5cd joi ns Cys138 to Cys143 while the other joins Cys166 to Cys406.