Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes.
Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding domain (Cel9A-68) followed by a fibronectin III-like domain and a family 2 cellulose binding domain. To study its catalytic mechanism, 12 mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned, and expressed in Escherichia coli. The purified mutant enzymes were assayed for their activities on (carboxymethyl)cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, and 2,4-dinitrophenyl beta-D-cellobioside. They were also tested for ligand binding, enzyme processivity, and thermostability. The results clearly show that E424 functions as the catalytic acid, D55 and D58 are both required for catalytic base activity, and Y206 plays an important role in binding, catalysis, and processivity, while Y318 plays an important role in binding of crystalline cellulose substrates and is required for processivity. Several amino acids located in a loop at the end of the catalytic cleft (T245-L251) were deleted from Cel9A-68, and this enzyme showed slightly improved filter paper activity and binding to BMCC but otherwise behaved like the wild-type enzyme. The FnIII-like domain was deleted from Cel9A-90, reducing BMCC activity to 43% of the wild type.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
Weilin
W
Zhou
Diana C
DC
Irwin
Jose
J
Escovar-Kousen
David B
DB
Wilson
D000192:Actinomycetales; D019943:Amino Acid Substitution / Q000235:genetics*; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D001665:Binding Sites / Q000235:genetics; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004789:Enzyme Activation / Q000235:genetics; D005353:Fibronectins / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D008239:Lysine / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017384:Sequence Deletion; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D001426:Bacterial Proteins; D005353:Fibronectins; D011994:Recombinant Proteins; D013912:Threonine; D001224:Aspartic Acid; D018698:Glutamic Acid; D014443:Tyrosine; D001120:Arginine; D002480:Cellulase; C451300:cel9A protein, Pseudomonas; D007930:Leucine; D008239:Lysine; D002266:Carboxymethylcellulose Sodium