J. Mol. Biol. (1996) 264, 337–349 Activity Studies and Crystal Structures of Catalytically Deficient Mutants of Cellobiohydrolase I from Trichoderma reesei Jerry Sta ˚ hlberg 1 , Christina Divne 1 , Anu Koivula 2 , Kathleen Piens 3 Marc Claeyssens 3 , Tuula T. Teeri 2 and T. Alwyn Jones 1 * 1 Department of Molecular The roles of the residues in the catalytic trio Glu212-Asp214-Glu217 in Biology, University of cellobiohydrolase I (CBHI) from Trichoderma reesei have been investigated by changing these residues to their isosteric amide counterparts. Three Uppsala, Biomedical Centre P.O. Box 590, S-751 24 mutants, E212Q, D214N and E217Q, were constructed and expressed in Uppsala, Sweden T. reesei . All three point mutations significantly impair the catalytic activity of the enzyme, although all retain some residual activity. On the small 2 VTT Biotechnology and Food chromophoric substrate CNP-Lac, the k cat values were reduced to 1/2000, Research, P.O. Box 1500 1/85 and 1/370 of the wild-type activity, respectively, whereas the K M FIN-02044 VTT, Espoo values remained essentially unchanged. On insoluble crystalline cellulose, Finland BMCC, no significant activity was detected for the E212Q and E217Q mutants, whereas the D214N mutant retained residual activity. The 3 Department of Biochemistry consequences of the individual mutations on the active-site structure Microbiology and Physiology were assessed for two of the mutants, E212Q and D214N, by X-ray Faculty of Science crystallography at 2.0 Å and 2.2 Å resolution, respectively. In addition, the University of Gent structure of E212Q CBHI in complex with the natural product, cellobiose, K.L. Ledeganckstraat 35 was determined at 2.0 Å resolution. The active-site structure of each B-9000 Gent, Belgium 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--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.  1996 Academic Press Limited Keywords: cellulase; cellobiohydrolase I; catalytic activity; active-site mutant; crystal structure *Corresponding author Introduction In nature, cellulose is present predominantly in the cell walls of land-based higher plants where it provides strength and form to the plant cells, but also the flexibility needed to allow cells to divide and grow. Cellulose itself is a chemically simple polymer, consisting of -1,4-linked D-glucosyl units; the structure of lignocellulose, however, is complex due to its composite nature. Although the cellulose-degrading machinery of the filamentous soft-rot fungus Trichoderma reesei has been studied extensively, the catalytic action undertaken by the individual enzymes remains poorly understood. Enzymatic action on the solid, highly crystalline substrate is facilitated by a multidomain structure found in most cellulolytic enzymes. The major components of the cellulolytic enzyme system of T. reesei are well adapted for hydrolysis at the surface boundary of the solid substrate, and most enzymes have a two-domain organization with a Abbreviations used: BMCC, bacterial micro-crystalline cellulose; CBD, cellulose-binding domain; CBHI, cellobiohydrolase I; CNP-Lac, 2-chloro-4-nitrophenyl--lactoside; EGI, endoglucanase I; EGZ, endoglucanase Z; HEC, hydroxyethyl cellulose; IBTG, o -iodobenzyl-1-thio--D-glucoside; NAG, N-acetyl glucosamine; NCS, non-crystallographic symmetry; PNPC, p-nitrophenyl -cellobioside; r.m.s., root-mean-square. 0022–2836/96/470337–13 $25.00/0  1996 Academic Press Limited