Effect of secondary substrate binding in penicillopepsin: contributions of subsites S3 and S2' to kcat.
The kinetic parameters kcat, KM, and kcat/KM were determined at 25 degrees C and pH 4.5, 5.5, and 6.0 for the series of penicillopepsin substrates Ac-Alam-Lys-(NO2)Phe-Alan-amide, where (NO2)Phe is p-nitrophenylalanine and m and n equal 0-3. KM values at pH 6.0 were the same for all 12 peptides and averaged 0.088 +/- 0.02 mM but increased to different degrees at lower pH. In contrast, kcat values increased with increasing chain length. At pH 6 and at the pH optimum of kcat, the largest increases (about 37-fold on average) were obtained when alanine residues were added in positions P2' and P3. Only 1-2-fold increases were observed for positions P2, P3', P4, and P4'. These results show that occupation of subsites S2' and S3 is largely responsible for the rate enhancements caused by secondary substrate interactions with this series of peptides. Additional support for an important role of subsite S3 comes from the observation that the two peptides where m = 1 and n = 1 or 2, respectively, are cleaved not only between lysine and p-nitrophenylalanine but also between the latter and alanine, suggesting that occupation of subsite S3 by the N-terminal alanine overcomes the unfavorable interaction of alanine in subsite P1'. Subsite S3 is also important in the binding of pepstatin analogues and in transpeptidation reactions. It is proposed that the roles of subsites S3 and S2' are to facilitate the conversion of the first enzyme-substrate complex into a productive complex and to assist in the distortion of the scissile bond.(ABSTRACT TRUNCATED AT 250 WORDS)
Hofmann
T
T
Department of Biochemistry, University of Toronto, Canada.
Allen
B
B
Bendiner
M
M
Blum
M
M
Cunningham
A
A
D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity
D007202:Indicators and Reagents; D009842:Oligopeptides; D010450:Endopeptidases; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases