[Thermitase--a thermostable serine protease. VIII. Kinetic and ESR investigations on the interactions of enzymes with spin labeled peptide methyl ketones].

An attempt was made to study the structure of the active center of thermitase by means of spin labeled peptide analogues. For this purpose, peptide methyl ketones SL-Alan-PheCH3 of different chain length (n = 0, 1, 2, 3; SL = 2,2,5,5-Tetramethyl-pyrrolin-1-oxyl-3-carbonyl) were synthesized. Synthesis and physico-chemical properties of these compounds are described and inhibition constants Ki for the interaction of these compounds with thermitase were measured. SL-Ala2-PheCH3 and SL-Ala3-PheCH3 are strong reversible inhibitors of thermitase with Ki values of 8.9 X 10(-6) M and 4.8 X 10(-7) M, respectively, whereas the analogous compounds with n = 0 and n = 1 represent only reduced affinity for this enzyme. ESR spectra of SL-Ala2-PheCH3 and SL-Ala3-PheCH3 in the presence of thermitase reveal that a great part of the SL residues of enzyme bound inhibitor molecules is not measurably restricted in their mobility whereas another part is hindered by unspecific interaction with the protein. The kinetic and ESR data are discussed with regard to the substrate binding region of the enzyme.