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
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
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DOI：
10.1074/jbc.271.20.11676
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发表时间：
1996-05-17
影响因子：
4.8
通讯作者：
Erneux, C
中科院分区：
文献类型：
--
作者：
Communi, D;Lecocq, R;Erneux, C
通过化学修饰和定点突变，研究了人I型D-肌醇-1，4，5-三磷酸(INS(1，4，5)P-3)5-磷酸酶底物结合域中两个反应性精氨酸残基的关键作用。苯乙醛对该酶的化学修饰伴随着对酶活性的不可逆抑制。我们的研究表明，苯乙二醛形成酶抑制复合体，在INS(1，4，5)P-3，D-肌醇1，3，4，5-四磷酸酯(INS(1，3，4，5)P-4)或2，3-二磷酸甘油酸酯(2，3-BPG)的存在下，修饰反应被阻止。直接与共价修饰酶结合的[H-3]Ins(1，4，5)P-3显著减少。C-14标记的苯乙醛的化学计量比为每摩尔酶掺入2.1摩尔的苯乙醛。放射性标记的INS(1，4，5)P-3 5-磷酸酶经α-胰凝乳酶蛋白降解和反相高效液相色谱分离得到单一的[C-14]苯乙醛修饰的多肽。该序列(M-N-T-R-C-P-A-W-C-D-R-I-L)与INS(1，4，5)P-3 5-磷酸酶的第340-352位氨基酸相对应。不同苯硫代海因氨基酸衍生物的放射性估计表明，修饰的氨基酸是Arg-343和Arg-350。此外，两个精氨酸残基通过定点突变获得了两个突变酶，两个突变酶具有相同的紫外圆二色谱(CD)。这两个突变体(即R343A和R350A)显示INS(1，4，5)P-3的K-m值增加(分别是10倍和15倍)，导致酶活性急剧下降。综上所述，我们已经直接鉴定出两个反应性精氨酸残基是INS(1，4，5)P-35-磷酸酶活性部位的一部分。这些结果表明，在肌醇和磷脂酰肌醇多聚5-磷酸酶的一级结构中保守的10个氨基酸的长序列片段在底物识别中起着至关重要的作用。
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-3) 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-3, D-myo-inositol 1,3,4,5-tetrakisphosphate (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 dramatically reduced. The stoichiometry of labeling with C-14-labeled phenylglyoxal is shown to be 2.1 mol of phenylglyoxal incorporated per mol of enzyme. A single [C-14]phenylglyoxal-modified peptide is isolated following 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) corresponds to amino acids 340-352 of Ins(1,4,5)P-3 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-m values for Ins(1,4,5)P-3 (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-3 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.