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The kinetics of calcium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by fluorescence stopped-flow methods, using the calcium indicator Quin 2. Two distinct rate proccsscs, each corresponding to the release of two calcium ions are resolved for calmodulin at both low and high ionic strength.The effect of 0.1 M KCI is to accelerate the slow process from 9.1 1.5 s f l to 24 k 6.0 s -l and to reduce the rate of the fast process from 650 s-l to 240 k 50 s -l at 25°C. In the presence of 0.1 M KCI it was possible to determine activation parameters for the fast process: A H # = 41 f 5 kJ mo1-l and A S # = -63 17 J K -' m o l -I . These values are in good agreement with those obtained by 43Ca NMR.Studies of the tryptic fragments TR 1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, clearly identified Ca2+-binding sites I and I 1 as thc low-affinity (rapidly dissociating) sites and sites 111 and 1V as the high-affinity (slowly dissociating) sites. The kinetic properties of the two proteolytic fragments are closely similar to the List and slowly dissociating sites of native calmodulin, supporting the idea that calmodulin is constructed from two largely independent domains.The presence of the calmodulin antagonist trifluoperazine markedly decreased the Ca2 ' dissociation rates from calmodulin. One of the two high-affinity trifluoperazine-binding sites was found to be located on the Nterminal half and the other on the C-terminal half of calmodulin. The affinity of the C-terminal site is at least one order of magnitude greater.

Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.z 1998 Federation of European Biochemical Societies.

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