D. Communi et al., ARGININE 343 AND 350 ARE 2 ACTIVE-SITE RESIDUES INVOLVED IN SUBSTRATE-BINDING BY HUMAN TYPE-I D-MYO-INOSITOL 1,4,5-TRISPHOSPHATE 5-PHOSPHATASE, The Journal of biological chemistry, 271(20), 1996, pp. 11676-11683
The crucial role of two reactive arginyl residues within the substrate
binding domain of human Type I D-myo-inositol 1,4,5-trisphosphate (In
s(1,4,5)P-3) 5-phosphatase has been investigated by chemical modificat
ion 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 t
he presence of either Ins(1,4,5)P-3, D-myo-inositol 1,3,4,5-tetrakisph
osphate (Ins(1,3,4,5)P-4) or 2,3-bisphosphoglycerate (2,3-BPG). Direct
[H-3]Ins(1,4,5)P-3 binding to the covalently modified enzyme is drama
tically reduced. The stoichiometry of labeling with C-14-labeled pheny
lglyoxal is shown to be 2.1 mol of phenylglyoxal incorporated per mol
of enzyme. A single [C-14]phenylglyoxal-modified peptide is isolated f
ollowing a-chymotrypsin proteolysis of the radiolabeled Ins(1,4,5)P-3
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) corresp
onds to amino acids 340-352 of Ins(1,4,5)P-3 5-phosphatase. An estimat
e 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 mutage
nesis 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-m values for Ins(1,4,5)P-3 (10- a
nd 15-fold, respectively) resulting in a dramatic loss in enzymic acti
vity. In conclusion, we have directly identified two reactive arginyl
residues as part of the active site of Ins(1,4,5)P-3 5-phosphatase. Th
ese results point out the crucial role for substrate recognition of a
10 amino acids-long sequence segment which is conserved among the prim
ary structure of inositol and phosphatidylinositol polyphosphate 5-pho
sphatases.