Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli.
Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3.6.1.1] from Escherichia coli Q13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.
Samejima
T
T
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Tokyo.
Tamagawa
Y
Y
Kondo
Y
Y
Hachimori
A
A
Kaji
H
H
Takeda
A
A
Shiroya
Y
Y
D001665:Binding Sites; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism; D006358:Hot Temperature; D043564:Inorganic Pyrophosphatase; D010084:Oxidation-Reduction; D010777:Photochemistry; D011755:Pyrophosphatases / Q000378:metabolism / Q000528:radiation effects; D012395:Rose Bengal; D013050:Spectrometry, Fluorescence; D013774:Tetranitromethane; D014443:Tyrosine / Q000378:metabolism
D012395:Rose Bengal; D014443:Tyrosine; D006639:Histidine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D013774:Tetranitromethane