Histidine 89 is an essential residue for Hsp70 in the phosphate transfer reaction.
Autophosphorylation of Hsp70 is detected in the process of substrate refolding in the presence of adenosine triphosphate (ATP) in the reaction mixture. But to date, the role and mechanism of Hsp70 autophosphorylation have not been elucidated. In this study we determined the site of histidine phosphorylation of Hsp70 as an intermediate in the process of phosphate transfer reaction by site-directed mutagenesis. We selected two possible sites (ie, His89 and His227) of intermediate histidine phosphorylation based on our hypothesis of the transfer of gamma-phosphoryl groups and replacement by glycine and serine. Although an acid labile autophosphorylation intermediate of Hsp70 and its cytidine diphosphate-dependent dephosphorylation were detected in wild-type Hsp70, they were markedly suppressed in the H89S mutation of Hsp70, but not on the H227S mutation. The ATPase activity and ATP synthesis activity of Hsp70 were almost completely suppressed in the H89S and H89G mutations. The role of His89 in the phosphate transfer reaction of Hsp70 is discussed.
Lu
Yuanming
Y
Department of Clinical Research Center, No. 6 Hospital, Shanghai Jiaotong University, Shanghai 200233, People's Republic of China. luym650920@hotmail.com
Hu
Qian
Q
Yang
Cuixia
C
Gao
Feng
F
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003565:Cytidine Diphosphate / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D050505:Mutant Proteins; D012694:Serine; D006639:Histidine; D000244:Adenosine Diphosphate; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D000251:Adenosine Triphosphatases; D005998:Glycine