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(m) 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.
Lee
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LV
Institute for Enzyme Research and Department of Biochemistry, University of Wisconsin, 1710 University Avenue, Madison, WI 53705, USA.
Gerratana
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B
Cleland
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WW
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010428:Pentosephosphates / Q000378:metabolism; D010429:Pentoses / Q000494:pharmacology; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D012255:Ribitol / Q000378:metabolism; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000378:metabolism
D010428:Pentosephosphates; D010429:Pentoses; D013402:Sugar Alcohols; C041025:ribitol-5-phosphate; D012255:Ribitol; C100182:ribulose; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022550:ribulokinase; D008274:Magnesium; C014999:arabitol