Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism.
Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii. They display 30% sequence identity. The binding constants of the E. coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes. They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate. The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1). The Km for Dha of both kinases is <6 microM. The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine. Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal. However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols. The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol.
Garcia-Alles
Luis F
LF
Department of Chemistry and Biochemistry and Laboratory for Chemical and Mineralogical Crystallography, University of Berne, CH-3012 Berne, Switzerland.
Siebold
Christian
C
Nyffeler
Therese Lüthi
TL
Flükiger-Brühwiler
Karin
K
Schneider
Philipp
P
Bürgi
Hans-Beat
HB
Baumann
Ulrich
U
Erni
Bernhard
B
D000096:Acetone / Q000031:analogs & derivatives* / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000588:Amines / Q000737:chemistry; D001667:Binding, Competitive; D016971:Citrobacter freundii / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004099:Dihydroxyacetone Phosphate / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005985:Glyceraldehyde / Q000737:chemistry; D007700:Kinetics; D010728:Phosphoenolpyruvate / Q000737:chemistry*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D011485:Protein Binding; D011789:Quantum Theory; D013379:Substrate Specificity
D000588:Amines; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D000096:Acetone; D005985:Glyceraldehyde; D004099:Dihydroxyacetone Phosphate; C006973:chloroacetone; D010728:Phosphoenolpyruvate; C004433:acetol; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase