Further Studies on the Interaction between a Protein Proteinase Inhibitor, Streptomyces Subtilisin Inhibitor, and Thiolsubtilisin BPN'1

Abstract

The interaction between Streptomyces subtilisin inhibitor (SSI) and thiolsubtilisin BPN', in which the catalytic residue Ser 221 of subtilisin BPN' [EC 3.4.21.14] is converted chemically to Cys 221, was examined and compared with the interaction between SSI and native subtilisin BPN'. SSI competitively inhibits the esterolytic activity of thiolsubtilisin BPN' toward p-nitrophenyl acetate with an inhibitor constant, K1 of 13 μM at pH 7.0, 25°C. An ultraviolet absorption difference spectrum having troughs around 245 nm and 295 nm was observed upon the binding of SSI and thiolsubtilisin BPN' at alkaline pH (pH 9.0-10.4), 25°C. This difference spectrum is similar in shape to that observed on the binding between SSI and subtilisin BPN', indicating the protonation of a tyrosyl residue upon the binding (Inouye, K., Tonomura, B., & Hiromi, K. (1975) abstracts of papers, 26th Forum on Protein Structure, Nagasaki pp. 117-120). Spectrophotometric titration of the difference spectra yielded a value for the dissociation constant of the SSI-thiolsubtilisin BPN' complex, Kd, of 40 μM at pH 9.2-10.2, 25°C. These K and Kd values are more than 10 times greater than those for the interaction between SSI and subtilisin BPN' (Inouye, K., Tonomura, B., Hiromi, K., Sato, S., & Murao, S. (1977) J. Biochem. 82, 961–967; Fujiwara, K., Inouye, K., Tonomura, B., Murao, S., & Tsuru, D. (1977) J. Biochem. 82, 125–130; Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. 84, 1195–1202). This shows that a small change induced in the active Site structure of subtilisin BPN' leads to a considerable decrease in its binding affinity (by about 30 kJ/mol (7 kcal/ mol) in free energy units) to SSI. The pH-dependence of the difference spectra observed on the binding of SSI and thiolsubtilisin BPN' at alkaline pH shows that a tyrosyl residue of the enzyme, which has been assigned to be either Tyr 104 or Tyr 217 (Lnouye, K., Tonomura, B., & Hiromi, K. (1976) Seikagaku (in Japanese) 48, 542; Inouye, K., Tonomura, B., & Hiromi, K. (1979) J. Biochem. 85, 1115–1126), shifts its pKa value from 9.7 to 10.2–10.3 on complex formation with SSL. The extent of this upward pKa-shift (△pKa =0.5–0.6) is considerably smaller than that observed on the interaction of SSI and subtilisin BPN' (pKa 9.7→>11.5; △pKa>1.8) (Inouye, K., Tonomura, B., & Hiromi, K. (1975) abstracts of papers, 26th Forum on Protein Structure, Nagasaki pp. 117–120). The large difference in △pKa suggests that a change in the van der Waals radius as small as 0.45 A due to the conversion of -OH to -SH leads to a larger increase in the distance between the tyrosyl residue of the enzyme and the carboxyl group of SSI.