2cel: Difference between revisions

The roles of the residues in the catalytic trio Glu212-Asp214-Glu217 in, cellobiohydrolase I (CBHI) from Trichoderma reesei have been investigated, by changing these residues to their isosteric amide counterparts. Three, mutants, E212Q, D214N and E217Q, were constructed and expressed in T., reesei. All three point mutations significantly impair the catalytic, activity of the enzyme, although all retain some residual activity. On the, small chromophoric substrate CNP-Lac, the kcat values were reduced to, 1/2000, 1/85 and 1/370 of the wild-type activity, respectively, whereas, the KM values remained essentially unchanged. On insoluble crystalline, cellulose, BMCC, no significant activity was detected for the E212Q and, E217Q mutants, whereas the D214N mutant retained residual activity. The, consequences of the individual mutations on the active-site structure were, assessed for two of the mutants, E212Q and D214N, by X-ray crystallography, at 2.0 A and 2.2 A resolution, respectively. In addition, the structure of, E212Q CBHI in complex with the natural product, cellobiose, was determined, at 2.0 A resolution. The active-site structure of each mutant is very, similar to that of the wild-type enzyme. In the absence of ligand, the, active site of the D214N mutant contains a calcium ion firmly bound to, Glu212, whereas that of E212Q does not. This supports our hypothesis that, Glu212 is the charged species during catalysis. As in the complex of, wild-type CBHI with bound o-iodobenzyl-1-thio-beta-D-glucoside, cellobiose, is bound to the two product sites in the complex with E212Q. However, the, binding of cellobiose differs from that of the glucoside in that the, cellobiose is shifted away from the trio of catalytic residues to interact, more intimately with a loop that is part of the outer wall of the active, site.

2CEL is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hypocrea_jecorina Hypocrea jecorina] with NAG and CA as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Cellulose_1,4-beta-cellobiosidase Cellulose 1,4-beta-cellobiosidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.91 3.2.1.91] Structure known Active Sites: CAA, CAB, CTA and CTB. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2CEL OCA].  

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