Disulfide arrangement and functional domains of beta-1,4-endoglucanse E5 from Thermomonospora fusca.
Thermomonospora fusca cellulase E5 contains six cysteine residues. The number and location of the disulfide bonds and the effect of reduction of the disulfides and modification of the resulting half-cystine residues on enzymatic activity were determined. No free sulfhydryl groups were found in E5. Reduction and subsequent labeling with iodoacetamide of E5 and of an enzymatically active 32-kDa proteolytic derivative of E5 (E5cd) showed that one of the three disulfides is accessible to reduction under nondenatured conditions while the other two are not accessible. Full reduction of the disulfides and complete carboxymethylation of the six cysteines decrease the specific activity of E5 on CMC by more than half, but reduction of only the exposed disulfide bond does not affect enzymatic activity or binding of E5 to cellulose. A 14-kDa proteolytic fragment of E5 containing 120 amino acids from the N-terminus of the protein was shown to bind to crystalline cellulose. This confirms earlier evidence that the cellulose binding domain of E5 is located at the N-terminus of the protein. This 14-kDa fragment contains the accessible disulfide bond involving Cys93 and Cys100. The location of the two disulfide bonds in the other fragment (E5cd) was determined by cleaving it with cyanogen bromide under conditions that left the disulfide bonds intact. The resulting peptides were separated under both nonreducing and reducing conditions using RP-HPLC. Amino acid analysis of peptide peaks indicated that one disulfide linkage in E5cd joins Cys138 to Cys143 while the other joins Cys166 to Cys406.
McGinnis
K
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Section of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853.
Wilson
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DB
D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003488:Cyanogen Bromide; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis
D004220:Disulfides; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D002482:Cellulose; D002480:Cellulase; D003545:Cysteine; D003488:Cyanogen Bromide; D004229:Dithiothreitol; D007460:Iodoacetamide