KINETIC CHARACTERIZATION OF UDP-GLUCOSE PYROPHOSPHORYLASE FROM GERMINATED BARLEY (MALT)

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 mu m and 228 U mg(-1) for glucose-1-phosph ate and 93 mu M and 255 U mg(-1) for UTP at pH 8.35. UTP gave a substr ate surplus inhibition with a K-iS of 7.09 mM. For the pyrophosphoryly sis 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 c ompetitive inhibitor (K-i 0.117 mM) for UTP and a non-competitive inhi bitor (0.615 mM) for glucose-1-phosphate. In the pyrophosphorylysis re action, UTP is a competitive inhibitor (0.169 mM) for UDP-glucose. Ino rganic 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 f or glucose-1-phosphate and UTP in the synthesis reaction. The analysis of these inhibition studies revealed a sequential ordered Bi-Bi mecha nism. The enzyme is inhibited by free UTP and the optimum ratio of Mg2 +/UTP for synthesis of UDP-glucose is between 5 and 10. The enzyme sho ws relative activities for CTP (18.5%), GTP (14.3%) and ATP (13.7%) wh en glucose-l-phosphate is the second substrate.