PHOSPHORYLATION OF MYCOBACTERIUM-LEPRAE HEAT-SHOCK-70-PROTEIN AT THREONINE-175 ALTERS ITS SUBSTRATE-BINDING CHARACTERISTICS

We have examined the functional properties including autophosphorylati on of the Mycobacterium leprae Hsp70 homologue. Recombinant M. leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphory lating 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. Autophosphorylatio n was augmented by up to 25 mM Ca2+. Using site-directed mutagenesis t o construct two Thr-->Ala mutants at positions 175 and 193, and phosph oamino acid analysis, it was shown that Thr(175) was the dominant thre onine residue autophosphorylated in M. leprae Hsp70. Phosphorylation l ed to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin. These properties are compared with thos e of the DnaK protein of Escherichia coli. (C) 1998 Elsevier Science B .V. All rights reserved.