Substrate-dependent activation of uridine diphosphoglucose dehydrogenase from the dimorphic fungus Mucor rouxii

Abstract

As part of an investigation on polyuronide synthesis in the dimorphic fungus Mucor rouxii, we have studied the synthesis from UDP-glucose (UDPG) of the activated sugar precursor UDPGlucuronic acid. UDPG dehydrogenases from yeastlike and mycelial forms of M. rouxii were partially purified by ammonium sulfate fractionation and ultrafiltration. The enzyme preparations were largely free of enzymes which could degrade the substrates or products of the reaction. In the assay, enzymes from both sources exhibited slow activation over a period of several minutes. The rate of activation was dependent on both the enzyme concentration and the concentration of UDPG and NAD. The enzyme from the mycelial form had a km for UDPG of 490 μ m, a K m for NAD of 297 μ m, and a molecular weight at pH 7.0 of 440,000 as estimated by gel chromatography. In contrast the enzyme from the yeastlike form had a km for UDPG of 168 μ m, a K m for NAD of 740 μ m, and a molecular weight at pH 7.0 of 330,000. At pH 8.0, both enzymes dissociated to give forms of lower molecular weight. At this pH, UDPG alone could activate the enzyme. This activation was accompanied by the association of the lower-molecular-weight forms to give the higher-molecular-weight forms seen at pH 7.0. Association was prevented by heat treatment. The heat-treated enzymes could not produce UDPGlucuronic acid.