Enzymes in organic synthesis: Use of subtilisin and a highly stable mutant derived from multiple site-specific mutations

Journal Article · Wed Jan 31 04:00:00 EST 1990 · Journal of the American Chemical Society; (USA)

DOI:https://doi.org/10.1021/ja00159a006· OSTI ID:6829678

Wong, Chi-Huey; Chen, S T; Hennen, W J; Bibbs, J A; Wang, Y F; Liu, J L.C. ^[1]; Pantoliano, M W; Whitlow, M; Bryan, P N ^[2]

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1. Texas A and M Univ., College Station (USA)
2. Genex Corp., Gaithersburg, MD (USA)

A subtilisin mutant (subtilisin 8350) derived from subtilisin BPN' via six site-specific mutations (Met50Phe, Gly169Ala, Asn76Asp, Gln206Cys, Tyr2l7Lys, and Asn2l8Ser) was found to be 100 times more stable than the wild-type enzyme in aqueous solution at room temperature and 50 times more stable than the wild type in anhydrous dimethylformamide. Kinetic studies using ester, thio ester, and amide substrates, and the transition-state analogue inhibitor Boc-Ala-Val-Phe-CF{sub 3}, indicate that both the wild-type and the mutant enzymes have very similar specificities and catalytic properties. The inhibition constant (Ki = 5.0 {mu}M) for the wild-type enzyme is approximately 5 times that of the mutant enzyme (Ki = 1.1 {mu}M), suggesting that the mutant enzyme binds the reaction transition state more strongly than the wild-type enzyme. This result is consistent with the observed rate constants for the corresponding ester and amide substrates; i.e., the k{sub cat}/K{sub m} values for the mutant are larger than those for the wild-type enzyme. Application of the mutant enzyme and the wild-type enzyme to organic synthesis has been demonstrated in the regioselective acylation of nucleosides in anhydrous dimethylformamide (with 65-100% regioselectivity at the 5'-position), in the enantioselective hydrolysis of N-protected and unprotected common and uncommon amino acid esters in water (with 85-98% enantioselectivity for the L-isomer), and in the synthesis of di- and oligopeptides via aminolysis of N-protected amino acid and peptide esters. The enzymatic peptide synthesis was carried out under high concentrations of DMF ({approximately}50%) to improve substrate solubility and to minimize enzymatic peptide cleavage. Low enantioselectivity was observed in the enzymatic transformation of non-amino acid alcohols and acids.

• DOE Contract Number: AC03-76SF00098
• OSTI ID: 6829678
• Journal Information: Journal of the American Chemical Society; (USA), Journal Name: Journal of the American Chemical Society; (USA) Vol. 112:3; ISSN 0002-7863; ISSN JACSA
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
400201 -- Chemical & Physicochemical Properties
550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BIOTECHNOLOGY
CATALYSIS
DATA
DATA ANALYSIS
ENZYME ACTIVITY
EXPERIMENTAL DATA
INFORMATION
KINETICS
MEASURING INSTRUMENTS
MEASURING METHODS
NUMERICAL DATA
ORGANIC COMPOUNDS
PEPTIDES
PROTEINS
REACTION KINETICS
SYNTHESIS