Substituted glycals as probes of glycosidase mechanisms

Abstract

d-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis β-glucosidase in order to probe structure/function relationships in this enzyme. d-Glucal is shown to be a substrate ( k cat = 2.3 min −1, K m = 0.85 mM) undergoing hydration with stereospecific protonation from the α-face to yield 2-deoxy-β- d-glucose. 1-Methyl- d-glucal surprisingly serves as only a poor substrate ( k cat = 0.056 min −1, K m = 57 mM), also undergoing protonation from the α-face. 2-Fluoro- d-glucal, however, is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak ( K i = 24 mM) inhibitor. Similar behaviour was seen with almond β-glucosidase and yeast α-glucosidase and for the interaction of 2-fluoro- d-galactal with Escherichia coli β-galactosidase. A series of of α,β-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis β-glucosidase. Of these only 1-nitro- d-glucal functioned as a time dependent, irreversible inactivator ( k i = 0.011 min −1, K i = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed mutliple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors ( K i = 3–96 mM).