Biochemical Journal

Research article

Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant

Dieter BECKER, Christophe BRAET, Harry BRUMER III, Marc CLAEYSSENS, Christina DIVNE, B. Richard FAGERSTRÖM, Mark HARRIS, T. Alwyn JONES, Gerard J. KLEYWEGT, Anu KOIVULA, Sabah MAHDI, Kathleen PIENS, Michael L. SINNOTT, Jerry STÅHLBERG, Tuula T. TEERI, Melanie UNDERWOOD, Gerd WOHLFAHRT


The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala224) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Å (= 0.1nm) closer to the acid/base Glu217 residue, with a 3.1 Å contact between N∊2 and O∊1. The pH variation of kcat/Km for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK1 shifting from 2.22±0.03 in the wild-type to 3.19±0.03 in the mutant, and pK2 shifting from 5.99±0.02 to 6.78±0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative kcat for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower kcat/Km values for both lactosides and cellobiosides, and a marginally lower stability. However, kcat values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

  • cellulase
  • cellulose
  • endoglucanase
  • enzyme kinetics
  • pH-dependence


  • Abbreviations used: BMCC, bacterial microcrystalline cellulose; CBH, cellobiohydrolase; CNP, 2-chloro-4-nitrophenyl; DNP, 3,4-dinitrophenyl; EG, endoglucanase; ES, enzyme–substrate; H. i., Humicola insolens; MU-Lac, 4-methylumbelliferyl β-d-lactoside; RMS, root mean square; T. r., Trichoderma reesei; wt, wild-type; E223S etc., Glu223 → Ser etc.; PDB, Protein Data Bank.