SSI-subtilisin BPN complex

Despite the large molecular weight, the, 3C NMR spectrum of [M]SSI-subtilisin BPN complex in an aqueous solution clearly showed three enhanced resonances in the carbonyl... 

Fig. 2.6 Assignment of the Met carbonyl signals of the SSI-subtilisin BPN complex by reverse double labeling methods. A) [M]SSI-subtilisin BPN complex B) [M, revl4N-V]SSI-subtiIisin BPN complex C) [M, rev14N-C]SSI-subtilisin BPN complex. (0.7 mM, 40°C, 50 mM phosphate D2O buffer pD=7.3,75.4 MHz). The result of the assignment of Met residues of [M]SSI-subtilisin BPN complex by this reverse labeling method was indicated on the spectrum (A).

We compared the l3C NMR spectra of the subtilisin complexes of [M]SSI and [M]SSI. Rather surprisingly, the NMR spectrum taken within two hours after the preparation of [M]SSI -subtilisin BPN complex was absolutely identical to that of [M]SSI-subtilisin BPN. A period of two hours was necessary to obtain the 13C NMR spectrum of the [M]SSI -subtilisin complex with a sufficient signal-to-noise ratio. This included the time after the modified inhibitor was brought in contact with subtilisin BPN. There were no extra signgals detected other than those observed for the [M]SSI-subtilisin complex, indicating that the cleaved scissile bond in [M]SSI can be rapidly restored in the complex. Since time-resolution of 13C NMR spectroscopy is rather limited by its inherent insensitivity, we are not able to tell exactly how fast this process is. Tonomura et al., however, have recently found by a stopped flow technique an unknown kinetic process having a half-life time of two seconds for the SSI -subtilisin system. Obviously this process should be the restoration process of the cleaved scissile bond of SSI in the complex. Therefore, the hydrolyzed scissile bond could in fact be restored within several seconds (private communication). 

The conformation of M73R SSI in the free state was nearly identical to that of the WT SSI since there was almost no chemical shift difference between these two inhibitors. The carbonyl carbon chemical shift profile of the M73R SSI-subtilisin BPN complex was also very similar to that of the WT SSI-subtilisin BPN complex, showing that the conformations of these complexes are also nearly the same. The shift profile of the M73R SSI-bovine trypsin, however, was quite different from those of the subtilisin complexes. This can be taken as a good indication that the conformational flexible segments are responsible for the wide inhibitory activity of SSI. 

Table 2.1 List of the chemical shifts of [M]SSI in free and complexed state with subtilisin BPN (40° C, pD=7.3). Complexation shift (c)=shift (b) - shift (a)...

It has been established that the complex between SSI and subtilisin BPN exists in aqueous solution as the Michaelis intermediate with the intact undistorted scissile bond. This intermediate seems to be identical to that found in the solid state, although the precise... 

Fig. 2.7 Differences in the chemical shift of the SSI backbone carbonyl l3C resonances in free and complexed state with subtilisin BPN (pH 7.3, 40° C). The dark and dotted bars show down- and up-field shifts by complexation, respectively. Small solid circles on the residue numbers (horizontal scale) represent the examined residues. The residues in direct contact with the complexed subtilisin are shown by small open circles. (Reproduced with permission from Y. Miyake, Thesis, Tokyo Metropolitan University (1992))...

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