Abstract
The crucial role of two reactive arginyl residues within the substrate binding domain of human Type I D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 5-phosphatase has been investigated by chemical modification and site-directed mutagenesis. Chemical modification of the enzyme by phenylglyoxal is accompanied by irreversible inhibition of enzymic activity. Our studies demonstrate that phenylglyoxal forms an enzyme-inhibitor complex and that the modification reaction is prevented in the presence of either Ins(1,4,5)P3, D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) or 2,3-bisphosphoglycerate (2,3-BPG). Direct [3H]Ins(1,4,5)P3 binding to the covalently modified enzyme is dramatically reduced. The stoichiometry of labeling with 14C-labeled phenylglyoxal is shown to be 2.1 mol of phenylglyoxal incorporated per mol of enzyme. A single [14C]phenylglyoxal-modified peptide is isolated following alpha-chymotrypsin proteolysis of the radiolabeled Ins(1,4,5)P3 5-phosphatase and reverse-phase high performance liquid chromatography (HPLC). The peptide sequence (i.e. M-N-T-R-C-P-A-W-C-D-R-I-L) corresponds to amino acids 340-352 of Ins(1,4,5)P3 5-phosphatase. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivatives shows the modified amino acids to be Arg-343 and Arg-350. Furthermore, two mutant enzymes were obtained by site-directed mutagenesis of the two arginyl residues to alanine, and both mutant enzymes have identical UV circular dichroism (CD) spectra. The two mutants (i.e. R343A and R350A) show increased Km values for Ins(l,4,5)P3 (10- and 15-fold, respectively) resulting in a dramatic loss in enzymic activity. In conclusion, we have directly identified two reactive arginyl residues as part of the active site of Ins(1,4,5)P3 5-phosphatase. These results point out the crucial role for substrate recognition of a 10 amino acids-long sequence segment which is conserved among the primary structure of inositol and phosphatidylinositol polyphosphate 5-phosphatases.
Highlights
Phate (Ins(1,4,5)P3)1 and 1,2-diacylglycerol are generated from phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by receptor-mediated activation of phospholipase C
Protection was provided by the other substrate Ins(1,3,4,5)P4, and the competitive inhibitor 2,3-BPG. These results suggest that at least one reactive arginyl residue is involved in substrate binding
We aimed to identify active site residues interacting with the substrate for human Type I InsP3 5-phosphatase
Summary
Phate (Ins(1,4,5)P3) and 1,2-diacylglycerol are generated from phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by receptor-mediated activation of phospholipase C (for review, see Refs. 1 and 2). Covalent and irreversible modification with amino acid specific reagents has been used successfully to identify lysyl or arginyl residues in the substrate binding domain in many enzymes, such as tyrocidine synthetase 1 (21), Ca2ϩ/ATPase (22), 6-phosphofructo-2-kinase (23), and Ins(1,4,5)P3 3-kinase (24). Two arginyl residues were shown using site-directed mutagenesis to be critical to bind the C-2 phospho group of fructose 2,6-bisphosphate in rat. We investigated the possibility that active site arginines may play such a role in substrate binding for Type I Ins(1,4,5)P3 5-phosphatase. We identified two essential arginines, i.e. Arg-343 and Arg-350, taking part of the sequence segment R-X-P-A-W-C-D-R-I-L This segment appears to be crucial for enzymic activity, especially for substrate binding. The data have been confirmed by site-directed mutagenesis of both arginyl residues This represents the first direct identification of an inositol polyphosphate 5-phosphatase active site peptide
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