Use of adenosine (5')polyphospho(5')pyridoxals to study the substrate-binding region of glutathione synthetase from Escherichia coli B.
Adenosine(5')polyphospho(5')pyridoxals (APn-PLs, n = 2, 3, 4) were examined for affinity labeling of glutathione synthetase (EC 6.3.2.3) from Escherichia coli B. When the enzyme was incubated with an APn-PL or pyridoxal phosphate in the presence of Mg2+ and then reduced with sodium borohydride, it was most rapidly inactivated by AP4-PL. AP4-PL had a high affinity to the enzyme. The dissociation constant of AP4-PL in the inactivation process was 23 microM. The enzyme was almost completely protected from inactivation by addition of either ATP or gamma-glutamylcysteine. Complete inactivation corresponded to the incorporation of 1 mol of AP4-PL/mol of subunit of the tetrameric enzyme. Proteolytic digestion and sequence analysis of the AP4-PL-labeled enzyme revealed that only Lys-18 was modified. In contrast, the less efficient AP3-PL was found attached to Lys-17, Lys-18, Lys-144, and Lys-148. In the three-dimensional structure of the enzyme, Lys-18 is located close to the putative gamma-glutamylcysteine-binding site, but Lys-17, Lys-144, and Lys-148 are in the mouth of the inner-solvent region, at the bottom of which is the active-site cleft. Furthermore, difference Fourier analysis with the AP4-PL-soaked crystal of the enzyme showed that the adenosine moiety of the bound AP4-PL was in the crevice, which is the ATP-binding site of the enzyme. These results demonstrate the bivalent binding of AP4-PL lying across the gamma-glutamylcysteine- and ATP-binding sites.
Hibi
T
T
Institute for Chemical Research, Kyoto University, Japan.
Kato
H
H
Nishioka
T
T
Oda
J
J
Yamaguchi
H
H
Katsube
Y
Y
Tanizawa
K
K
Fukui
T
T
D000227:Adenine Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D000345:Affinity Labels / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D005981:Glutathione Synthase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*
D000227:Adenine Nucleotides; D000345:Affinity Labels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D005981:Glutathione Synthase; D008274:Magnesium