Truncated variants of Serratia proteamaculans oligopeptidase B having different activities

• Published in: Biochemistry (Moscow) 2015-10, Vol.80 (10), p.1331-1343
• Main Authors: Mikhailova, A. G., Nekrasov, A. N., Zinchenko, A. A., Rakitina, T. V., Korzhenevsky, D. A., Lipkin, A. V., Razguljaeva, O. A., Ovchinnikova, M. V., Gorlenko, V. A., Rumsh, L. D.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Binding Sites; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Caseins - chemistry; Caseins - metabolism; Chymotrypsin - chemistry; Chymotrypsin - metabolism; Enzymatic activity; Enzymes; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Eukaryotes; Genetic aspects; Genetic variation; Glass - chemistry; Gram-positive bacteria; Hydrolysis; Life Sciences; Mass spectrometry; Microbiology; Molecular Sequence Data; Molecular Weight; Nocardia corallina; Observations; Peptides; Physiological aspects; Proteases; Proteins; Proteolysis; Sequence Deletion; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Serratia - enzymology; Trypsin - chemistry; Trypsin - metabolism
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297915100156

Treatment of native psychrophilic oligopeptidase B from Serratia proteamaculans (PSP, 78 kDa) with chymotrypsin (soluble or immobilized on modified porous glass MPG-PA) in the presence of 50% glycerol leads to production of a truncated enzyme form (PSP-Chtr, ∼66 kDa), which retains activity toward the low molecular weight substrate of PSP, BAPNA, but in contrast to PSP, is active toward the protein substrate azocasein. It has been shown by MALDI-TOF massspectrometry that PSP-Chtr lacks the N-terminal region of the molecule that envelops the catalytic domain of PSP and supposedly prevents hydrolysis of high molecular weight substrates. It has also been established that the lacking fragment corresponds to the N -terminal highest rank element of the informational structure of PSP. This finding confirms the usefulness of the method of informational structure analysis for protein engineering of enzymes. A similar treatment of PSP with immobilized trypsin also led to production of a stable truncated enzyme form (PSP-Tr, ∼75 kDa) which lacked 22 C -terminal amino acid residues and completely lost enzymatic activity, presumably because of changes in the nearest environment of His652 of the catalytic triad.