A Single Calcium Binding Site Is Crucial for the Calcium-Dependent Thermal Stability of Thermolysin-like Proteases

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• 作者：Oene R. Veltman ; Gert Vriend ; Herman J. C. Berendsen ; Bertus Van den Burg ; Gerard Venema ; and Vincent G. H. Eijsink
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：April 14, 1998
• 年：1998
• 卷：37
• 期：15
• 页码：5312 - 5319
• 全文大小：484K
• 年卷期：v.37,no.15(April 14, 1998)
• ISSN：1520-4995

文摘

Thermostable thermolysin-like proteases (TLPs), such as the TLP ofBacillus stearothermophilusCU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single(Ca3, Ca4) calcium binding sites.The single sites are absent in thermolabile TLPs, suggesting thatthey are determinants of (variation in)TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-steindeed reduced thermal stability, but onlymutations in the Ca3 site affected the calcium-dependence of stability.The predominant effect of theCa3 site results from the fact that the Ca3 site is part of a region ofTLP-ste, which unfolding is crucialfor thermal inactivation. Thermal inactivation is not caused bythe absence of calcium from the Ca3 siteper se, but rather by unfolding of a region of TLP-ste for whichstability depends on the occupancy of theCa3 site. In accordance with this concept is the observation thatthe effects of mutations in the Ca3 sitecould be compensated by stabilizing mutations near this site. Inaddition, it was observed that thecontribution of calcium binding to the Ca3 was substantially reduced inextremely stable TLP-ste variantscontaining multiple stabilizing mutations in the Ca3 region.Apparently, in these latter variants, unfoldingof the Ca3 region contributes little to the overall process of thermalinactivation.