Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase

L-Ribulokinase is unusual among kinases since it phosphorylates all four 2-ketopentoses with almost the same k(cat) values. The K-m's differ, however, being 0.14 mM for L- and 0.39 mM for D-ribulose and 3.4 mM for L- and 16 mM for D-xylulose. In addition, L-arabitol is phosphorylated at C-5 (K 4 mM) and ribitol (adonitol) is phosphorylated to D-ribitol-5-phosphate (K-m 5.5 mM), but D-arabitol, xylitol, and aldopentoses are not substrates. The K-m's for MgATP depend on the substrates, being 0.02 mM with L-ribulose, 0.027 mM with D-ribulose and L-xylulose, and 0.3-0.5 mM with the other substrates. In the absence of a sugar substrate there is an ATPase with K-m of 7 mM and k(cat) 1% of that with sugar substrates. The initial velocity pattern is intersecting, and MgAMPPNP is competitive vs MgATP and uncompetitive VS L-ribulose. L-Erythrulose is competitive VS L-ribulose and when MgATP concentration is varied induces substrate inhibition which is partial. These data show that the mechanism is random, but there is a high level of synergism in the binding of sugar and MgATP, and the path in which the sugar adds first is strongly preferred. (C) 2001 Elsevier Science .