The relevance of low-affinity Ca-binding sites to the structure, stability and kinetic properties of thermolysin

Abstract

To clarify the significance of low-affinity Ca-binding sites in thermolysin (TLN), we compared the enzymes having bound and free weak Ca-binding sites. Binding measurements suggested that TLN comprises two strong and two weak Ca-binding sites. Far-UV CD analysis indicated that the weak sites do not affect TLN’s secondary structure. However, a slight local rearrangement due to the weak sites’ occupancy was deduced by the fluorescence measurements. Upon Ca-binding to weak sites, K _M and k _cat for hydrolysis of 3-(-2-furylacryloyl)-l-glycyl-l-leucine-amide were reduced about 2.8- and 1.8-fold, respectively, and k _cat/K _M was therefore improved about 1.5-fold. Our modeling studies suggest that the modified kinetic properties may be due to altered hinge angle between N- and C-terminal domains upon Ca-binding to the weak sites. TLN exhibited a higher temperature optimum, remarkable increase in the half-life of thermoinactivation t _1/2 and enhanced remaining activity in the presence of denaturant agents in a Ca-dependent manner. In contrast, organic solvents and pH stabilities were irrelevant to the weak sites. Finally, we conclude that low-affinity sites of TLN may induce subtle changes in the local environment and modulate the enzymatic activity and thermal stability.