HYPERTHERMOSTABLE MUTANTS OF BACILLUS-LICHENIFORMIS ALPHA-AMYLASE - THERMODYNAMIC STUDIES AND STRUCTURAL INTERPRETATION

This paper provides further understanding of the thermodynamic and str uctural features determining the stability of Bacillus licheniformis a lpha-amylase (BLA) at two crucial positions, His133 and Ala209. Result s of protein modelling and saturated site-directed mutagenesis at posi tion 133 and 209 have been reported in a previous paper (Declerck ef a l., 1995, Prot. Engng, 8, 1029-1037). In the first part of the present work, evidence is presented supporting the hypothesis that the stabil izing mutations reduce the rate of initial unfolding of the enzyme dur ing the reversible step of the inactivation reaction and do not modify the irreversible processes undergone subsequently by the unfolded mol ecules. In the second part, we have examined the three-dimensional str ucture of BLA which has been determined recently by X-ray analysis (Ma chius et al., 1995, J. Mol. Biol., 246, 545-559). This analysis showed that our previous predictions made from molecular modelling were part ly correct. At position 209, the effect of the stabilizing substitutio ns can be explained by a groove-filling effect reinforcing the hydroph obic packing between two helices of the central domain, while preservi ng a well-ordered water structure at the surface. At position 133, the stabilizing substitutions must compensate the loss of the hydrogen bo nd network in which the original histidine side-chain is involved; thi s compensation could be achieved through enhanced hydrophobic side-cha in interactions within the P-sheet where residue 133 is located, which correlates with the propensity of the residue to form and maintain a beta-strand conformation of the main chain at this position.