Kim Dae Won, Hong Joung Woo, Eum Won Sik, Choi Hee Soon, Choi Soo Hyun, Kim So Young, Lee Byung Ryong, An Jae Jin, Lee Sun Hwa, Lee Seung Ree, Kwon Oh-Shin, Kwon Hyeok Yil, Cho Sung-Woo, Lee Kil Soo, Park Jinseu, Choi Soo Young
Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
J Biochem Mol Biol. 2005 Jan 31;38(1):58-64. doi: 10.5483/bmbrep.2005.38.1.058.
Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.
肌醇单磷酸酶（IMPP）是磷酸肌醇细胞信号系统中的关键酶。本研究发现，将来自猪脑的IMPP与磷酸吡哆醛（PLP）一起孵育会导致酶活性随时间而失活。光谱证据表明，失活是通过与酶的氨基形成席夫碱来进行的。在用硼氢化钠还原失活的酶后，观察到每摩尔酶二聚体中有1.8摩尔磷酸吡哆醛残基被掺入。底物肌醇-1-磷酸可保护该酶免受PLP的失活作用。在用PLP修饰该酶后进行胰蛋白酶消化，通过反相高效液相色谱法分离出了一个在210nm处有吸收的放射性肽段。该肽段的氨基酸测序确定了PLP结合位点的一部分是包含序列L-Q-V-S-Q-Q-E-D-I-T-X的区域，其中X表示无法确定的苯硫基乙内酰脲氨基酸。然而，该肽段的氨基酸组成结果表明，缺失的残基可能被指定为磷酸吡哆醛赖氨酸。这表明IMPP的催化功能是通过PLP与蛋白质底物结合位点处或附近的特定赖氨酰残基结合来调节的。
