Regulation and mutational analysis of the HPr kinase/phosphorylase from Bacillus subtilis.
In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the HPr kinase/phosphorylase (HprK/P). It has recently been shown that HprK/P could catalyze the phosphorylation of the protein HPr by using pyrophosphate (PP(i)) as a phosphate donor instead of ATP. Here we showed that, as for ATP, PP(i) binds to the enzyme with strong positive cooperativity. However, in contrast to ATP, PP(i) binding does not modify the fluorescence properties of the unique Trp residue of Bacillus subtilis HprK/P. In addition, to understand how two conserved motifs, namely, the P-loop and the specific signature of this family, participate in the three enzymatic activities of HprK/Ps (ATP-kinase, PP(i)-kinase, and phosphorylase), several site-directed mutants were generated. Whereas the three activities are mediated by the P-loop which is directly involved in the binding of ATP, PP(i), or Pi, the signature motif seems to be involved preferentially in the dephosphorylation reaction. On the basis of these results, we propose a model in which the binding of the allosteric activator FBP induces a conformational change of a central loop located above the active site of HprK/P, thereby allowing the ATP binding. However, this conformational change is not required for the binding of PP(i).
Pompeo
Frédérique
F
Laboratoire de Chimie Bactérienne, UPR 9043, IBSM-CNRS, 31 chemin Joseph Aiguier, 13402 Marseille, France.
Granet
Yohann
Y
Lavergne
Jean-Pierre
JP
Grangeasse
Christophe
C
Nessler
Sylvie
S
Jault
Jean-Michel
JM
Galinier
Anne
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer; D005635:Fructosediphosphates / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D005635:Fructosediphosphates; D010710:Phosphates; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D008274:Magnesium; C029063:fructose-1,6-diphosphate