Inactivation of brain myo-inositol monophosphate phosphatase by pyridoxal-5'-phosphate.
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.
Kim
Dae Won
DW
Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
Hong
Joung Woo
JW
Eum
Won Sik
WS
Choi
Hee Soon
HS
Choi
Soo Hyun
SH
Kim
So Young
SY
Lee
Byung Ryong
BR
An
Jae Jin
JJ
Lee
Sun Hwa
SH
Lee
Seung Ree
SR
Kwon
Oh-Shin
OS
Kwon
Hyeok Yil
HY
Cho
Sung-Woo
SW
Lee
Kil Soo
KS
Park
Jinseu
J
Choi
Soo Young
SY
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D007295:Inositol Phosphates / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism; D010669:Phenylthiohydantoin / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology*; D013552:Swine; D013997:Time Factors
D000596:Amino Acids; D001894:Borohydrides; D007295:Inositol Phosphates; D010446:Peptide Fragments; C002647:inositol 1-phosphate; D010669:Phenylthiohydantoin; D011732:Pyridoxal Phosphate; C025364:sodium borohydride; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D008239:Lysine