Rate studies of polysaccharide biosynthesis from guanosine diphosphate α-d-glucose and guanosine diphosphate α-d-mannose

Abstract

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of 14C-labelled polysaccharide formation from GDP-α-d-[14C]glucose is not increased by additions of GDP-α-d-mannose. However, final incorporation is increased by addition of GDP-α-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose is increased by all concentrations of GDP-α-d-glucose that are less than that of the GDP-α-d-[14C]mannose. Maximum stimulation of the initial rate occurs at a GDP-α-d-[14C]mannose/GDP-α-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-α-d-[14C]mannose is decreased by the addition of GDP-α-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2–3 and 50μm. Extrapolated Km values for the two linear components are 0.4–1.0 and 700–1500μm. The effect of GDP-α-d-glucose on the kinetics of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. 14C-labelled polysaccharide formation from GDP-α-d-[14C]glucose also results in biphasic reciprocal plots. One component appears to have Km about 2–3μm, the other about 200–400μm. In this reaction, GDP-α-d-mannose appears to be a competitive inhibitor with Ki 20–30μm. With particulate preparations of P. aureus, GDP-α-d-[14C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-α-d-[14C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.