Interaction of subtilisin BPN' and recombinant Streptomyces subtilisin inhibitors with substituted P1 site residues.
Kinetic analysis was performed on the interaction between subtilisin BPN' and recombinant species of a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), of which the P1 site amino acid residue, Met73, was replaced by site-directed mutagenesis. The inhibitor constant, Ki, was determined from the residual enzyme activity by using a peptide substrate. The rate constant of binding, kon, and the rate constant of dissociation, koff, were determined from a progress curve of the substrate hydrolysis in the presence of the inhibitor by using newly derived equations. A recombinant SSI in which Met73 was replaced by Ile showed an affinity (1/Ki) toward subtilisin BPN' of only about 7% of that of the wild-type SSI, and the kinetic analysis revealed that the increase of koff was responsible for this difference. The affinity of other SSI mutants in which Met73 was replaced by Glu or Asp decreased significantly as pH became increasingly alkaline. The decrease in the affinity of these recombinants was due to the decrease of kon rather than the increase of koff. Stopped-flow studies revealed that the binding reaction was reconcilable with a two-step mechanism, and the kinetic parameters for each step were obtained for the binding of the enzyme and recombinant SSIs.
Masuda-Momma
K
K
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University.
Hatanaka
T
T
Inouye
K
K
Kanaori
K
K
Tamura
A
A
Akasaka
K
K
Kojima
S
S
Kumagai
I
I
Miura
K
K
Tonomura
B
B
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013381:Subtilisins / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C018150:subtilisin inhibitor protein, Streptomyces; D013381:Subtilisins