STRUCTURAL IDENTIFICATION OF THE MYOINOSITOL 1,4,5-TRISPHOSPHATE-BINDING DOMAIN IN RAT-BRAIN INOSITOL 1,4,5-TRISPHOSPHATE 8-KINASE

A series of key amino acids involved in Ins(1,4,5)P-3 (InsP(3)) bindin g and catalytic activity of rat brain InsP(3) 3-kinase has been identi fied. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 2 80, 125-129]. In this study, newly prepared 5'-deletion and site-direc ted mutants have been compared both for InsP(3) binding and InsP(3) 3- kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP(3) binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP(3 ) binding, and this finding suggests that these residues (i.e. (LDCK26 2)-D-259) involved in InsP(3) binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C2 61 and K262. The two new enzymes were designated M4 (C261S) and M5 (K2 62A). M4 showed similar V-max and K-m values for InsP(3) and ATP to wi ld-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin-Sepharose. C-terminal deletion mutants t hat had lost five, seven or nine amino acids showed a large decrease i n InsP(3) binding and InsP(3) 3-kinase activity. One mutant that had l ost five amino acids (M2) was purified to apparent homogeneity: K-m va lues for both substrates appeared unchanged but V-max was decreased ap prox. 40-fold compared mas with the wild-type enzyme. The results indi cate that (1) a positively charged amino acid residue K262 is essentia l for InsP(3) binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP(3) 3-kinase re action.