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J. Biochem, 1988, Vol. 103, No. 5 766-772
© 1988 Japanese Biochemical Society

research-article

Chemical Modifications of Histidyl and Tyrosyl Residues of Inorganic Pyrophosphatase from Escherichia coli

Tatsuya Samejima, Faculty of Textile Science and Technology*,1, Yuko Tamagawa*,2, Yoshifusa Kondo*, Akira Hachimori**, Hiroyuki Kaji*, Atsushi Takeda*** and Yoko Shiroya*,3

* Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University Setagaya-ku, Tokyo 157
** Institute of High Polymer Research,Shinshu University Ueda, Nagano 386
*** Department of Clinical Pathology, Showa University, Fujigaoka Hospital, Showa University School of Medicine Midori-ku, Yokohama, Kanagawa 227

1To whom correspondence should be addressed.

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, imidodi-phosphate 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.

2Present address: Department of Drug Metabolism and Product Development, Nippon Roche Research Center, Kamakura, Kanagawa 247.

3Present address: Biological Laboratory, Wayo Women's University, Ichikawa, Chiba 272.


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