The interaction of 1-fluoro-D-glucopyranosyl fluoride with glucosidases.
1. 1-Fluoro-D-glucopyranosyl fluoride undergoes pH-independent loss of F- ion at a rate of 1 x 10(-8) s-1 at 50.0 degrees C, some 10(3)-fold slower than alpha-D-glucopyranosyl fluoride and 4 x 10(4)-fold slower than beta-D-glucopyranosyl fluoride. 2. The (inverting) amyloglucosidase II of Aspergillus niger hydrolyses the difluoride according to Michaelis-Menten kinetics (Km 34 mM and kcat. 0.27 s-1), by apparently the same (simple) mechanism by which it hydrolyses alpha-D-glucopyranosyl fluoride (Km 38 mM and kcat. 730 s-1), rather than by the Hehre resynthesis-hydrolysis mechanism used to transform beta-D-glucopyranosyl fluoride. 3. The difluoride is also a substrate for the (inverting) trehalase of pig kidney [Km 17.3 mM and Vmax. 6.2 x 10(-4) relative to alpha-D-glucopyranosyl fluoride (Km 38 mM]). 4. The quantitatively similar effect of fluorine substitution on the one-step enzymic reactions and on the non-enzymic reactions suggests that they go through similar (oxocarbonium-ion-like) transition states. 5. The difluoride is a substrate for the (retaining) beta-glucosidases from Aspergillus wentii (A3 enzyme) and sweet-almond meal (B isoenzyme) and for the retaining alpha-glucosidase from rice: comparison with the appropriate monofluoride reveals a variable rate-retarding effect of the second fluorine atom on kcat./Km that correlates with other measures of oxocarbonium ion character in the transition state. 6. The difluoride is a substrate for the (retaining) alpha-glucosidase from yeast, but also gives an insidious mimicry of active-site-directed irreversible inhibition, which we tentatively attribute either to formation of the non-covalent complex or to the fluoroglucosyl-enzyme increasing the well-known tendency of this enzyme to come out of solution by adsorption on the walls of the vessel.
Konstantinidis
A
A
Department of Organic Chemistry, University of Bristol, U.K.
Sinnott
M L
ML
D000818:Animals; D001230:Aspergillus / Q000201:enzymology; D001234:Aspergillus niger / Q000201:enzymology; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism; D005947:Glucose / Q000031:analogs & derivatives* / Q000378:metabolism; D005959:Glucosidases / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D012275:Oryza / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine; D014198:Trehalase / Q000378:metabolism; D000520:alpha-Glucosidases / Q000378:metabolism; D001617:beta-Glucosidase / Q000378:metabolism
D007527:Isoenzymes; C071645:1-fluoroglucopyranosyl fluoride; D005959:Glucosidases; D000520:alpha-Glucosidases; D001617:beta-Glucosidase; D014198:Trehalase; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose