Cation-Binding Sites of Subtilisin Carlsberg Probed with Eu(III) Luminescence

AbstractTwo Ca2+-binding sites of subtilisin Carlsberg are studied by monitoring static and time-resolved luminescence of selectively substituted Eu3+ at each site, and they are found to be characteristically quite different from each other. Compared with the coordination sphere of free Eu3+, two sites are very similar to each other, so that both have a well-defined binding structure with low coordination symmetry. However, compared with the weak site, the strong site is relatively more polar, more symmetrical, and more easily accessible. Furthermore, despite the absence of water reported in the x-ray crystal structure (Bode et al., 1987, Eur. J. Biochem. 166:673–692), one water molecule is found to exist in the coordination sphere of the strong site in aqueous solution. Thus it is suggested that in solution the Ca2+ bound in the strong site forms an additional coordination bond to a solvent or substrate molecule