HPr kinase/phosphorylase (HPrK/P) is the key regulator of carbon metabolism in many Gram-positive bacteria. It phosphorylates/dephosphorylates the HPr protein of the bacterial phosphotransferase system on a regulatory serine residue in response to the nutrient status of the cell. In Mycoplasma pneumoniae, HPrK/P is one of the very few regulatory proteins encoded in the genome. The regulation of this enzyme by metabolites is unique among HPrK/P proteins studied so far: it is active as a kinase at low ATP concentrations, whereas the proteins from other bacteria need high ATP concentrations as an indicator of a good nutrient supply for kinase activity. We studied the interaction of M. pneumoniae HPrK/P with ATP, Fru1,6P2 and Pi by fluorescence spectroscopy. In agreement with the previously observed unique regulation, we found a very high affinity for ATP (K(d)=5.4 microM) compared with the HPrK/P proteins from other bacteria. The Kd for Fru1,6P2 was three orders of magnitude higher, which explains why Fru1,6P2 has only a weak regulatory effect on M. pneumoniae HPrK/P. Mutations of two important regions in the active site of HPrK/P, the nucleotide binding P-loop and the HPrK/P family signature sequence, had different effects. P-loop region mutations strongly affect ATP binding and thus all enzymatic functions, whereas the signature sequence motif seems to be important for the catalytic mechanism rather than for nucleotide binding.