Rate studies of polysaccharide biosynthesis from guanosine diphosphate -D-glucose and guanosine diphosphate -D-mannose.

Abstract

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose is not increased by additions of GDP-alpha-d-mannose. However, final incorporation is increased by addition of GDP-alpha-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is increased by all concentrations of GDP-alpha-d-glucose that are less than that of the GDP-alpha-d-[(14)C]mannose. Maximum stimulation of the initial rate occurs at a GDP-alpha-d-[(14)C]mannose/GDP-alpha-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-alpha-d-[(14)C]mannose is decreased by the addition of GDP-alpha-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]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 50mum. Extrapolated K(m) values for the two linear components are 0.4-1.0 and 700-1500mum. The effect of GDP-alpha-d-glucose on the kinetics of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose also results in biphasic reciprocal plots. One component appears to have K(m) about 2-3mum, the other about 200-400mum. In this reaction, GDP-alpha-d-mannose appears to be a competitive inhibitor with K(i) 20-30mum. With particulate preparations of P. aureus, GDP-alpha-d-[(14)C]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-alpha-d-[(14)C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.