STRUCTURAL DETERMINANTS OF THE THERMOSTABILITY OF THERMOLYSIN-LIKE BACILLUS NEUTRAL PROTEASES

Abstract The neutral protease of B. stearothermophilus (NP-ste) is 13 degrees less thermostable than thermolysin, the neutral protease of B. thermoproteolyticus. The sequences of these two proteins differ at 45 positions. By introducing site-directed mutations in NP-ste, it was shown that only a few of these sequence differences account for the total difference in thermostability. All critical residues appeared to be located at the surface of the molecule, mainly in the 63–69 region, and no drastic mutational effects were observed at buried positions. A series of additional mutations was designed that confirmed this trend: mutations in the hydrophobic core of the protein showed only marginal effects, whereas Ala Pro mutations at positions 63, 65, 66 and 69 had pronounced effects on the stability of the enzyme. The results support a model in which it is assumed that local unfolding processes at the surface of the protein, which render the NP susceptible towards autolysis, determine the rate of thermal inactivation. Within the framework of this model mutations that have large effects on stability are expected to be located in regions that unfold relatively easily and that play a role in the early steps of global unfolding. The present results indicate that the 63–69 area in the N-terminal domain of NP-ste is such an early unfolding region.