General acid base catalysis of .alpha.-glucan phosphorylases: stereospecific glucosyl transfer from D-glucal is a pyridoxal 5'-phosphate and orthophosphate (arsenate) dependent reaction

Abstract

D-Glucal, containing a highly reactive double bond, can replace glucose 1-phosphate as the glucosyl donor in phosphorylase-catalyzed glucosyl transfer to a suitable oligo- or polysaccharide acceptor: D-glucal + Pi + (glucose)n .**GRAPHIC**. 2-deoxy-.alpha.-D-glucosyl(glucose)n .dblarw. 2-deoxy-.alpha.-D-glucose-1-P + (glucose)n. This reaction is catalyzed by .alpha.-glucan phosphorylases from rabbit skeletal muscle, potato tuber, and Escherichia coli. D-Glucal is only measurably consumed by .alpha.-glucan phosphorylases when Pi or arsenate is present. With saturating concentrations of these anions and a glucosyl acceptor, the D-glucal reaction proceeds at rates comparable with the rates of glucosyl transfer from glucose 1-phosphate and of phosphorolysis or arsenolysis of poly- or oligosaccharides. Furthermore, for the reaction to proceed, the enzyme must be in the active conformation containing the cofactor pyridoxal 5''-phosphate in its dianionic form. On the basis of 1H NMR spectra, it is proposed that protonation at C-2 of D-glucal gives rise to a hypothetical 2-deoxy-.beta.-D-glucose intermediate, yielding as a final product (2-deoxy-.alpha.-D-[2(e)-2H]glucose)n .alpha.(1.fwdarw. 4) saccharides. These 2-deoxy-.alpha.-D-glucose oligo- or polysaccharides are degraded by .alpha.-glucan phosphorylases by phosphorolysis or arsenolysis like natural linear and branched .alpha.-glucans. The absolute requirement of the D-glucal reaction for phosphate (or arsenate) and its dependency on the dianionic form of the pyridoxal 5''-phosphate bound to phosphorylase are rationalized in terms of a proton transfer relay involving juxtaposed phosphates. Phosphate-phosphate interactions were postulated by Withers et al.