Active site-directed inhibition by optically pure epoxyalkyl cellobiosides reveals differences in active site geometry of two 1,3-1,4-beta-D-glucan 4-glucanohydrolases. The importance of epoxide stereochemistry for enzyme inactivation

• Published in: The Journal of biological chemistry Vol. 266; no. 18; pp. 11628 - 11631
• Main Authors: HØJ, P. B, RODRIGUEZ, E. B, ISER, J. R, STICK, R. V, STONE, B. A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.06.1991
• Subjects: 1,3-1,4- beta -D-glucan 4-glucanohydrolase; Analytical, structural and metabolic biochemistry; Bacillus subtilis - enzymology; Binding Sites; Biological and medical sciences; Cellobiose - analogs & derivatives; Cellobiose - pharmacology; Enzymes and enzyme inhibitors; Epoxy Compounds - chemistry; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - antagonists & inhibitors; Glycoside Hydrolases - metabolism; Hordeum - enzymology; Hydrolases; inhibitors; Isoenzymes - antagonists & inhibitors; Isoenzymes - metabolism; Protein Conformation; Stereoisomerism; Barley; Bacillales; Enzyme; Bacillus subtilis; Isozyme; Chain length; Aglycone; Enzyme inhibitor; Lichenase; Bacteria; Stereospecificity; Bacillaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)99003-4

1,3-1,4-beta-D-Glucan 4-glucanohydrolases (EC 3.2.-1.73) from Bacillus subtilis and barley (Hordeum vulgare) with identical substrate specificities but unrelated primary structures have been probed with (R,S)-epoxyalkyl (-propyl, -butyl, -pentyl) beta-cellobiosides and with optically pure (3S)- and (3R)-3,4-cellobiosides as active site-directed inhibitors. The optimal aglycon length for inactivation differs for the two enzymes, and they are differentially inhibited by the pure epoxybutyl beta-cellobioside diastereoisomers. The (3S)-epoxybutyl beta-cellobioside inactivates the B. subtilis enzyme much more efficiently than does the (3R)-isomer, whereas the reverse is true for the barley enzyme. Both enzymes are inactivated by a mixture of the stereoisomers at a rate intermediate of that observed with the individual isomers. The two beta-glucan endohydrolases may therefore employ sterically different mechanisms to achieve glycoside bond hydrolysis in their common substrate. The efficiency and specificity of epoxide-based "suicide" inhibitors may be enhanced significantly by the use of inhibitors bearing only one stereoisomeric form of the epoxide group.