Modification of a single tryptophan of the inorganic pyrophosphatase from thermophilic bacterium PS-3: possible involvement in its substrate binding.
The effect of N-bromosuccinimide (NBS) on the activity of the inorganic pyrophosphatase (PPiase) from thermophilic bacterium PS-3 was studied. The enzyme was almost completely inactivated on chemical modification with NBS, depending upon the concentration of NBS. The presence of a complex of Mg2+ and a substrate analogue, imidodiphosphate (PNP), provided extensive protection against the inactivation, whereas Mg2+ or PNP alone showed no protective effect. Amino acid analysis of the NBS-modified enzyme after hydrolysis with 6 M HCl indicated no change in the amino acid composition. However, the magnetic circular dichroism (MCD) bands around 293 nm due to the tryptophan residue and the optical density at 280 nm, decreased concomitantly with modification by NBS. These results strongly suggested that the tryptophan residue at position 143, which is the only tryptophan residue per subunit in the thermophilic PPiase (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A. (1990) J. Biochem. 108, 572-578), might be involved in the active site or be located in the vicinity of the active site. The circular dichroism (CD) spectrum in the far ultraviolet region showed no significant alteration during the modification, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modification considerably altered the CD bands in, the near ultraviolet region, indicating that a conformational change occurred in the vicinity of the active site in the enzyme molecule.
Kaneko
S
S
Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan.
Ichiba
T
T
Hirano
N
N
Hachimori
A
A
D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D002942:Circular Dichroism; D008274:Magnesium / Q000494:pharmacology; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000032:analysis*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism*
D014364:Tryptophan; D011755:Pyrophosphatases; D008274:Magnesium; D001974:Bromosuccinimide