title: Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics.
abstract: We have examined the functional properties including autophosphorylation of the Mycobacterium leprae Hsp70 homologue. Recombinant M. leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphorylating activities which were near pH 8 and 6, respectively. Both these activities were inhibited by reduced and alkylated bovine pancreatic trypsin inhibitor, but not other tested substrates. Autophosphorylation was augmented by up to 25 mM Ca2+. Using site-directed mutagenesis to construct two Thr-->Ala mutants at positions 175 and 193, and phosphoamino acid analysis, it was shown that Thr175 was the dominant threonine residue autophosphorylated in M. leprae Hsp70. Phosphorylation led to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin. These properties are compared with those of the DnaK protein of Escherichia coli.
authors.affiliation: Centenary Institute for Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW 2042, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Peake
authors.forename: N
authors.initials: N
authors.lastname: Winter
authors.forename: W
authors.initials: W
authors.lastname: Britton
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D007611:Aprotinin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009166:Mycobacterium leprae / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism
chemical_list: D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D010769:Phosphothreonine; D007611:Aprotinin; D000251:Adenosine Triphosphatases; D002118:Calcium