Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 8-kinase

摘要

A series of key amino acids involved in Ins(1,4,5)P-3 (InsP(3)) binding and catalytic activity of rat brain InsP(3) 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125-129]. In this study, newly prepared 5'-deletion and site-directed 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. (LDCK262)-D-259) involved in InsP(3) binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar V-max and K-m values for InsP(3) and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin-Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP(3) binding and InsP(3) 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: K-m values for both substrates appeared unchanged but V-max was decreased approx. 40-fold compared mas with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential 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 reaction.