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 mis 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 Cys(61)-Cys(90). An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp(51) to Arg(64) in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics, The Delta Delta G(U) 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 Delta G(U) 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 K-m correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (k(cat)/K-m) 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 K-m > 2-fold and k(cat) < 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 k(cat)), but it is slightly less stable.