1egn
|
CELLOBIOHYDROLASE CEL7A (E223S, A224H, L225V, T226A, D262G) MUTANT
OverviewOverview
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, (Ala(224)) 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 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a, 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of, k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately, bell-shaped for both wild-type and mutant, with pK(1) shifting from, 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2), 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, k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The, shift in the mutant pH optimum was associated with lower k(cat)/K(m), values for both lactosides and cellobiosides, and a marginally lower, stability. However, k(cat) 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.
About this StructureAbout this Structure
1EGN is a Single protein structure of sequence from Hypocrea jecorina with NAG and CO as ligands. Active as Cellulose 1,4-beta-cellobiosidase, with EC number 3.2.1.91 Full crystallographic information is available from OCA.
ReferenceReference
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., Becker D, Braet C, Brumer H 3rd, Claeyssens M, Divne C, Fagerstrom BR, Harris M, Jones TA, Kleywegt GJ, Koivula A, Mahdi S, Piens K, Sinnott ML, Stahlberg J, Teeri TT, Underwood M, Wohlfahrt G, Biochem J. 2001 May 15;356(Pt 1):19-30. PMID:11336632
Page seeded by OCA on Tue Nov 20 13:59:23 2007