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 μM for Dha, 780 μM for Dha-phosphate (DhaP), 50 μM for d,l-glyceraldehyde (GA), and 90 μM 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 K m for Dha of both kinases is