Binding between thermolysin and talopeptin (MKI) in which the tryptophan residue was converted into kynurenine.
The tryptophan residue of talopeptin, which is a specific inhibitor for thermolysin, was converted into kynurenine by ozonization followed by acid-catalyzed hydrolysis, and (Trp leads to Kyn) talopeptin (Kyn-talopeptin) thus obtained was purified with gel-chromatography. The inhibitor constant of Kyn-talopeptin, K1, and the dissociation constant of thermolysin-Kyn-talopeptin complex, Kd, directly obtained by fluorometric titration were in good agreement with each other. These values were found to be about 10 times larger than those of intact talopeptin, but both inhibitors showed a similar pH dependence. Upon the binding of Kyn-talopeptin with thermolysin, the protein fluorescence of thermolysin decreases by about 60%, while the kynurenine fluorescence (measured at 450 nm when excited at 360 nm) of the inhibitor increases about 14 times. The measurements of the excitation and fluorescence spectra of EI complex strongly indicated the energy transfer from tryptophan residue(s) (the donor) of the enzyme to kynurenine residue (the acceptor) of the inhibitor. The distance between the donor and the acceptor was roughly estimated to be 18 A. This value is in good agreement with the one expected from the crystallography of phosphoramidon-thermolysin complex. The binding process was studied kinetically with the stopped-flow method over the pH range between 4.5 and 8.5, by monitoring the decrease in the fluorescence intensity of the enzyme tryptophan caused by the complex formation. Comparison of the data with those previously obtained for talopeptin-thermolysin system revealed that the replacement of the tryptophan residue by kynurenine of the inhibitor does not affect the apparent second-order association rate constant, kon, seriously.
D001711:Biotransformation; D004734:Energy Metabolism; D006020:Glycopeptides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007737:Kynurenine / Q000096:biosynthesis*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013820:Thermolysin / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D006020:Glycopeptides; D007737:Kynurenine; C037819:talopeptin; D014364:Tryptophan; D013820:Thermolysin