Kinetic characterization of UDP-glucose pyrophosphorylase from germinated barley (malt)

Abstract

A complete data set of the kinetics and some features referring to the substrate specificity and inhibition of UDP-glucose pyrophosphorylase from germinated barley (malt) are given in order to evaluate the use of this easily available enzyme for the synthesis of nucleotide sugars in enzymic glycoconjugate synthesis. In the synthesis direction the K m and V max values were 74 μm and 228 U mg −1 for glucose-1-phosphate and 93 μM and 255 U mg −1 for UTP at pH 8.35. UTP gave a substrate surplus inhibition with a K iS of 7.09 mM. For the pyrophosphorylysis reaction the kinetic constants K m and V max were 0.191 mM and 350 U mg −1 for UDP-glucose and 0.172 mM and 345 U mg −1 for inorganic pyrophosphate. Inhibition studies demonstrated that UDP-glucose is a competitive inhibitor ( K i 0.117 mM) for UTP and a non-competitive inhibitor (0.015 mM) for glucose-1-phosphate. In the pyrophosphorylysis reaction, UTP is a competitive inhibitor (0.169 mM) for UDP-glucose. Inorganic pyrophosphate ( K i 0.213 and 0.952 mM) and inorganic phosphate ( K i 12.2 and 10.9 mM) were identified as non-competitive inhibitors for glucose-1-phosphate and UTP in the synthesis reaction. The analysis of these inhibition studies revealed a sequential ordered Bi-Bi mechanism. The enzyme is inhibited by free UTP and the optimum ratio of Mg 2+/UTP for synthesis of UDP-glucose is between 5 and 10. The enzyme shows relative activities for CTP (18.5%), GTP (14.3%) and ATP (13.7%) when glucose-1-phosphate is the second substrate.