Mutational analysis of the major loop of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases. Effects on protein stability and substrate binding.
The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1, 4-beta-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta-strand-(87-95) of the minor beta-sheet III of the protein core by a single disulfide bond at Cys61-Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The DeltaDeltaGU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 +/- 0.2 kcal.mol-1 for the disulfide bridge to protein stability. A good correlation is observed between DeltaGU values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.
Pons
J
J
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain.
Querol
E
E
Planas
A
A
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics*; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000235:genetics*; D013379:Substrate Specificity
D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; C028020:licheninase