Cation-binding sites of subtilisin Carlsberg probed with Eu(III) luminescence

Two Ca2+-binding sites of subtilisin Carlsberg are studied by monitoring st atic and time-resolved luminescence of selectively substituted Eu3+ at each site, and they are found to be characteristically quite different from eac h 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 struct ure with low coordination symmetry. However, compared with the weak site, t he 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, fur. J. Biochem. 166:673-692), one w ater molecule is found to exist in the coordination sphere of the strong si te in aqueous solution. Thus it is suggested that in solution the Ca2+ boun d in the strong site forms an additional coordination bond to a solvent or substrate molecule.