Arginine 343 and 350 Are Two Active Site Residues Involved in Substrate Binding by Human Type I D-myo-Inositol 1,4,5-Trisphosphate 5-Phosphatase

doi:10.1074/jbc.271.20.11676

Arginine 343 and 350 Are Two Active Site Residues Involved in Substrate Binding by Human Type I D-myo-Inositol 1,4,5-Trisphosphate 5-Phosphatase (*)

From the Institute of Interdisciplinary Research, University of Brussels, Campus Erasme, Building C, Route de Lennik 808, B-1070 Brussels, Belgium

^§ To whom correspondence should be addressed:
Institute of Interdisciplinary Research, University of Brussels, Campus Erasme, Bldg C, Route de Lennik 808, B-1070 Brussels, Belgium.
Tel.: 322-555-4164; Fax: 322-555-4655.

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)P) 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)P, D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1, 3,4,5)P) or 2,3-bisphosphoglycerate (2,3-BPG). Direct [³H]Ins(1,4,5)P 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 α-chymotrypsin proteolysis of the radiolabeled Ins(1,4,5)P 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)P 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 K values for Ins(1,4,5)P (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)P 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.

Footnotes

↵* This work was supported by grants of the FRSM, Boehringer Ingelheim, and the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Federal Service for Science, Technology and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Ins(1,4,5)P

D-myo-inositol 1,4,5-trisphosphate

Ins(1,3,4,5)P

D-myo-inositol 1,3,4,5-tetrakisphosphate

PtdIns(4,5)P

phosphatidylinositol 4,5-bisphosphate

PtdIns(3,4,5)P

phosphatidylinositol 3,4,5-trisphosphate

2,3-BPG

2,3-bisphosphoglycerate

Ni-NTA

nickel-nitrilotriacetic acid

HPLC

high performance liquid chromatography

PCR

polymerase chain reaction.
- Received January 19, 1996.
- Revision received February 15, 1996.