CLONING AND SEQUENCING OF 2 ENTEROCOCCAL GLPK GENES AND REGULATION OFTHE ENCODED GLYCEROL KINASES BY PHOSPHOENOLPYRUVATE-DEPENDENT, PHOSPHOTRANSFERASE SYSTEM-CATALYZED PHOSPHORYLATION OF A SINGLE HISTIDYL RESIDUE

The glpK genes of Enterococcus casseliflavus and Enterococcus faecalis , encoding glycerol kinase, the key enzyme of glycerol uptake and meta bolism in bacteria, have been cloned and sequenced, The translated ami no acid sequences exhibit strong homology to the amino acid sequences of other bacterial glycerol kinases, After expression of the enterococ cal glpK genes in Escherichia coli, both glycerol kinases were purifie d and were found to be phosphorylated by enzyme I and the histidine-co ntaining protein of the phosphoenolpyruvate: glycose phosphotransferas e system. Phosphoenolpyruvate-dependent phosphorylation caused a 9-fol d increase in enzyme activity, The site of phosphorylation in glycerol kinase of E. casseliflavus was determined as His-232, Site-specific m utagenesis was used to replace His-232 in glycerol kinase of E. cassel iflavus with an alanyl, glutamate, or arginyl residue. The mutant prot eins could no longer be phosphorylated confirming that His-232 of E. c asseliflavus glycerol kinase represents the site of phosphorylation. T he His(232) --> Arg glycerol kinase exhibited an about 3-fold elevated activity compared with wild-type glycerol kinase, Fructose 1,6-bispho sphate was found to inhibit E. casseliflavus glycerol kinase activity, However, neither EIIA(Glc) from E. coli nor the EIIA(Glc) domain of B acillus subtilis had an inhibitory effect on glycerol kinase of E. cas seliflavus.