ALPHA-1,4-GLUCAN LYASE, A NEW CLASS OF STARCH GLYCOGEN DEGRADING ENZYME .3. SUBSTRATE-SPECIFICITY, MODE OF ACTION, AND CLEAVAGE MECHANISM

The alpha-1,4-glucan lyase (EC 4.2.2.-), purified from the red alga Gr acilariopsis lemaneiformis, is a single polypeptide with a molecular m ass of 116654 Da as determined by matrix-assisted laser-desorption mas s spectrometry. It degraded maltose, maltosaccharides, amylose, amylop ectin and glycogen, forming 1,5-anhydro-D-fructose from the non-reduci ng end groups. The substrate specificity, mode of action, and cleavage mechanism of the enzyme were studied by using various naturally occur ring and synthesized substrates. This enzyme was highly specific for t he alpha-1,4-D-glucosidic bond. When a linear alpha-1,4-glucan was use d as substrate, the enzyme split the substrate from the non-reducing e nd and released 1,5-anhydro-D-fructose successively until only one glu cose unit was left. When a branched pentasaccharide of 6(2)-alpha-malt osylmaltotriose, obtained from glycogen by alpha-amylase Limitation, w as used as substrate, the glucose group in the 4-position of the 4,6-b ranched residue was not cleaved off. Using maltoheptaose as substrate and following the reaction with HPLC and H-1-NMR spectroscopy, it was found that the action mode of the lyase followed a multichain attack m echanism. H-1- and C-13-NMR spectroscopic studies on unlabelled and la belled amylose (1-H-2, 2-H-2, 1-C-13) as substrates indicated that the lyase cleaved the C-(1')-O(4) bond forming a double bond between C-1' and C-2', thus forming the enol form of 1,5-anhydro-D-fructose. It al so indicated that the catalytic process of the lyase involved proton e xchanges among C-1, C-2, C-3 and the solvent.