|Biely, Peter - SLOVAK ACADEMY|
|Mastihubova, Maria - SLOVAK ACADEMY|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 13, 2001
Publication Date: N/A
Technical Abstract: Acetylxylan esterase (AcXE) from Streptomyces lividans, which belongs to family 4 of carbohydrate esterases, exhibits unique catalytic properties and behaves as substrate-specific esterase. Based on deacetylation kinetics of methyl beta-D-xylopyranoside diacetates, it has been hypothesized that deacetylation of position 2 and 3, when neighboring position 3 and 2 are non-acetylated, involves an enzyme-catalyzed formatio of a five-membered ortho ester intermediate. To support the hypothesis, 2-deoxy-3,4-di-O-acetyl, 3-deoxy-2,4-di-O-acetyl, and 3-deoxy-3-fluoro- 2,4-di-O-acetyl methyl beta-D-xylopyranosides were synthesized and used as substrate analogues in which the formation of the ortho-ester intermediate is excluded. All three newly prepared derivatives were found to be extremely poor substrates for AcXE. The absence of OH-groups at position 2 or 3 impeded the reaction by orders of magnitude. The deacetylation was not inhibited by methyl beta-D-xylopyranoside, implying that the OH-groups at position 2 or 3 do not play any role in the binding of the substrate to the enzyme. With derivatives modified at position 3, the deacetylation took place at position 2. Two monoacetates were generated from the 2-deoxy-derivative. Experimental evidence and theoretical considerations support the previously proposed reaction mechanism. The proposed ortho-ester intermediate is similar to that proposed for aminoacyl-tRNA synthases.