Identification of amino acid residues responsible for the changes of absorption and fluorescence spectra on the binding of subtilisin BPN' and Streptomyces subtilisin inhibitor.
An ultraviolet absorption difference spectrum characteristic of the ionization change of a tyrosyl residue was observed on the binding of subtilisin BPN' with Streptomyces subtilisin inhibitor (SSI) at alkaline pH. This difference spectrum was considered to be induced by a pKa shift (from 9.7 to > or = 11.5) of a tyrosyl residue of subtilisin BPN' in the interaction with carboxyls of SSI [Inouye et al. (1979) J. Biochem. 85, 1115-1126]. In the present paper, the tyrosyl residue in subtilisin BPN' and the carboxyls in SSI were identified by analyzing the difference spectrum using mutants of subtilisin BPN' and SSI: naturally occurring mutants and those prepared by site-directed and cassette mutagenesis. The difference spectrum disappeared on the binding of a mutant subtilisin BPN' of which Tyr104 was replaced by Phe (S-BPN'Y104F) and SSI at pH 9.8. The magnitude of the absorption difference was much smaller when subtilisin BPN' was bound with a mutant SSI of which both Glu67 and Asp68 were replaced by Gly than with the wild-type SSI. These lines of evidence indicated that the difference spectrum was caused by Tyr104 of subtilisin BPN' interacting with Glu67 and Asp68 of SSI. The binding of subtilisin BPN' and SSI is accompanied by an increase of tryptophan fluorescence, which is pH-dependent in the range of pH 7-11 [Uehara et al. (1978) J. Biochem. 84, 1195-1202]. In the present study, this pH-dependence of the fluorescence diminished when SSI bound with S-BPN'Y104F. This suggested that the fluorescence increase was due to Trp106 of subtilisin BPN' and was influenced by the ionization of Tyr104.
Masuda-Momma
K
K
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University.
Shimakawa
T
T
Inouye
K
K
Hiromi
K
K
Kojima
S
S
Kumagai
I
I
Miura
K
K
Tonomura
B
B
D000596:Amino Acids / Q000737:chemistry*; D001407:Bacillus / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013381:Subtilisins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D001426:Bacterial Proteins; D046911:Macromolecular Substances; C018150:subtilisin inhibitor protein, Streptomyces; D013381:Subtilisins