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 Bacillus stearothermophilus by the double mutation G8C/N60C had resulted in an extremely thermostable enzyme with a half-life of 35.9 min at 92.5 degrees C [Mansfeld, J., Vriend, G., Dijkstra, B.W., Veltman, O.R., van den Burg, B., Venema, G., Ulbrich-Hofmann, R. & Eijsink, V.G. (1997) J. Biol. Chem. 272, 11152-11156]. The study in guanidine hydrochloride of this enzyme and the respective wild-type enzyme allowed us to distinguish between the stability toward global unfolding and autoproteolysis. At low protease concentrations (20 microg.mL-1) and short periods of incubation with guanidine hydrochloride (5 min), transition curves without the interference by autoproteolysis could be derived from fluorescence emission measurements. The effect of the disulfide bond on the global unfolding of the protein proved to be smaller than expected. In contrast, the measurement of autoproteolysis at higher protein concentrations (100 microg.mL-1) by quantitative evaluation of the bands of intact protein on SDS/PAGE revealed a strong stabilization toward autoproteolytic 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 the protease against autoproteolysis more than against global unfolding. Autoproteolysis starts as soon as the cleavage sites in flexible external structural regions become accessible. It is suggested that the stabilizing effect of the disulfide bond is caused by the fixation of the crucial loop region 56-69 or by hindrance of the primary cleavage in this region by the amino acid exchanges.
Dürrschmidt
P
P
Martin-Luther University Halle-Wittenberg, Department of Biochemistry/Biotechnology, Halle/Saale, Federal Republic of Germany.
Mansfeld
J
J
Ulbrich-Hofmann
R
R
D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D019791:Guanidine / Q000737:chemistry; D008958:Models, Molecular; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D013050:Spectrometry, Fluorescence
D004220:Disulfides; D011480:Protease Inhibitors; D010450:Endopeptidases; D019791:Guanidine