Regulation and Mutational Analysis of the HPr Kinase/Phosphorylase from Bacillus subtilis

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• 作者：Fré ; dé ; rique Pompeo ; Yohann Granet ; Jean-Pierre Lavergne ; Christophe Grangeasse ; Sylvie Nessler ; Jean-Michel Jault ; and Anne Galinier
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：June 10, 2003
• 年：2003
• 卷：42
• 期：22
• 页码：6762 - 6771
• 全文大小：243K
• 年卷期：v.42,no.22(June 10, 2003)
• ISSN：1520-4995

文摘

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 thephosphorylation 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 ofBacillus subtilis HprK/P. In addition, to understand how two conserved motifs, namely, the P-loop andthe 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 activitiesare mediated by the P-loop which is directly involved in the binding of ATP, PP_i, or Pi, the signaturemotif 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 changeof 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.