P. Peake et al., PHOSPHORYLATION OF MYCOBACTERIUM-LEPRAE HEAT-SHOCK-70-PROTEIN AT THREONINE-175 ALTERS ITS SUBSTRATE-BINDING CHARACTERISTICS, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1387(1-2), 1998, pp. 387-394
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.