Disulfide arrangement and chemical modification of beta-1,4-endoglucanase E2 from Thermomonospora fusca.
Thermomonospora fusca endoglucanase E2 contains six cysteine residues scattered along the protein sequence. Four of the cysteine residues were shown to participate in two disulfide bonds while the last two form a third disulfide bond. Neither full reduction of the disulfides nor complete carboxymethylation of all six cysteines totally destroys enzymatic activity, but the activity of the reduced enzyme is much lower than the native enzyme and the iodoacetamide-modified enzyme has very low activity. Reduction of only the accessible disulfides drastically decreases the enzyme's thermostability. One disulfide linkage joins Cys80 to Cys125, another joins Cys232 to Cys267, and the third joins Cys315 to Cys407. The first two bonds are similar to those in cellobiohydrolase II, which also belongs to cellulase family B (Rouvinen et al., 1990; Lao et al., 1991; Henrissat et al., 1989). Direct evidence for the involvement of carboxyl groups in catalysis by E2 was demonstrated by chemical modification with carbodiimide.
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; D002475:Cellobiose / Q000494:pharmacology; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002482:Cellulose / Q000494:pharmacology; D003488:Cyanogen Bromide; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D006358:Hot Temperature; D007460:Iodoacetamide / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011489:Protein Denaturation
D004220:Disulfides; D002475:Cellobiose; D002482:Cellulose; D002480:Cellulase; D003545:Cysteine; D003488:Cyanogen Bromide; D005022:Ethyldimethylaminopropyl Carbodiimide; D004229:Dithiothreitol; D007460:Iodoacetamide