Differentiation between conformational and autoproteolytic stability of the neutral protease from Bacillus stearothermophilus containing an engineered disulfide bond

The introduction of a disulfide bond into the neutral protease from Bacillu s stearothermophilus by the double mutation G8C/N60C had resulted in an ext remely thermostable enzyme with a half-life of 35.9 min at 92.5 degreesC [M ansfeld, J., Vriend, G., Dijkstra, B.W., Veltman, O.R., van den Burg, B., V enema, G., Ulbrich-Hofmann, R. & Eijsink, V.G. (1997) J. Biol. Chem. 272, 1 1152-11156]. The study in guanidine hydrochloride of this enzyme and the re spective wild-type enzyme allowed us to distinguish between the stability t oward global unfolding and autoproteolysis. At low protease concentrations (20 mug.mL(-1)) and short periods of incubation with guanidine hydrochlorid e (5 min), transition curves without the interference by autoproteolysis co uld be derived from fluorescence emission measurements. The effect of the d isulfide bond on the global unfolding of the protein proved to be smaller t han expected. In contrast, the measurement of autoproteolysis at higher pro tein concentrations (100 mug.mL(-1)) by quantitative evaluation of the band s of intact protein on SDS/PAGE revealed a strong stabilization toward auto proteolytic degradation by the disulfide bond. The rate of autoproteolysis in guanidine hydrochloride was found to be much lower than that of thermal denaturation, which can be attributed to the inhibition of the proteases by this denaturant. The results suggest that the disulfide bond stabilizes th e protease against autoproteolysis more than against global unfolding. Auto proteolysis starts as soon as the cleavage sites in flexible external struc tural regions become accessible. It is suggested that the stabilizing effec t of the disulfide bond is caused by the fixation of the crucial loop regio n 56-69 or by hindrance of the primary cleavage in this region by the amino acid exchanges.