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 kcat values. The Kmʹs 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 (Km 4 mM) and ribitol (adonitol) is phosphorylated to d-ribitol-5-phosphate (Km 5.5 mM), but d-arabitol, xylitol, and aldopentoses are not substrates. The Kmʹs 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 Km of 7 mM and kcat 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.