Identification of residues essential for catalysis and binding of calmodulin in rat brain inositol 1,4,5-trisphosphate 3-kinase.
In order to identify the amino acid residues involved in calmodulin (CaM) binding and catalytic activity, rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion protein [clone C5; Takazawa, Vandekerckhove, Dumont & Erneux (1990) Biochem. J. 272, 107-112]. Three deletion mutants in the plasmid of clone C5 were generated using convenient restriction enzymes. The results show that the removal of 34 amino acids from the C-terminal end of InsP3 3-kinase resulted in an inactive protein which still interacted with CaM-Sepharose in a Ca2(+)-dependent way. The catalytic domain is thus located at the C-terminal end of the protein. A series of 5' deletion mutants was prepared and used to produce proteins with the same C-terminal end but shortened N-termini, varying in length by over 80 amino acids. Assay of InsP3 3-kinase activity in bacterial extracts indicated that a maximum of 275 amino acids in the C-terminal region may be sufficient for the construction of a catalytically active domain. Affinity chromatography on CaM-Sepharose of 5' and 3' deletion mutants revealed that the sequence stretching from Ser-156 to Leu-189 is involved in CaM binding and enzyme stimulation.
Takazawa
K
K
Institut de Recherche Interdisciplinaire (IRIBHN), Université Libre de Bruxelles, Belgium.
Erneux
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000378:metabolism*; D002384:Catalysis; D002872:Chromosome Deletion; D015723:Gene Library; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D002147:Calmodulin; D011994:Recombinant Proteins; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase