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Volume 272, Number 20, Issue of May 16, 1997 pp. 13006-13012
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Mutational Analysis of the Major Loop of Bacillus 1,3-1,4--D-Glucan 4-Glucanohydrolases
EFFECTS ON PROTEIN STABILITY AND SUBSTRATE BINDING

(Received for publication, September 17, 1996, and in revised form, January 30, 1997)

Jaume Pons ^§ , Enrique Querol ^§ and Antoni Planas

From the  Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain and the ^§ Institut de Biologia Fonamental V. Villar Palasí and the Department de Bioquímica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1,4--D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to -strand-(87-95) of the minor -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 G_U between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 ± 0.2 kcal·mol¹ for the disulfide bridge to protein stability. A good correlation is observed between 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.

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