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

Abstract

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.

Dissociation of Hexameric Escherichia coli Inorganic Pyrophosphatase into Trimers on His-136 → Gln or His-140 → Gln Substitution and Its Effect on Enzyme Catalytic Properties

“…Our work suggests that this loss of activity is due to an effect on K m,h . Finally, by analogy with our present results, we speculate that the loss of E. coli PPase activity found by Samejima et al (1988) on chemical modification of His residues arises from dissociation of hexamer into trimers.…”

“…Earlier chemical modification studies of Samejima et al (1988) had implicated histidines as being involved in the activity of E. coli PPase. The absence of His residues from the active site cavity (Kankare et al, 1994) and the lack of conserved His residues in soluble PPases (Cooperman et al, 1992) make it clear that His residues have no direct role in catalysis, leaving open the possibility that the chemical modification results arise from an indirect effect.…”

Sructure ofEscherichia coliInorganic Pyrophosphatase at 2.2 Å Resolution

“…Based on chemical-modification experiments, histidines had been suggested to be important for catalytic activity (Hirano, Ichiba & Hachimori, 1991;Samejima et al, 1988) and so each histidine was substituted with glutamine . There are no conserved histidines (Cooperman et al, 1992); there are no histidines in the active site (Kankare et al, 1994); and no histidine is important for catalytic activity.…”

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus

“…These include histidine and tyrosine residues in E-PPase (Samejima et al, 1988) and B-PPase (Shiroya & Samejima, 1985), tryptophan in P-PPase (Kaneko et al, 1991) and E-PPase (Kaneko et al, 1993), serine (Avaeva et al, 1970), methionine (Yano et a l . , 1973), tryptophan (Negi et al, 1972), and histidine (Nazarova et al, 1972;Cooperman & Chiu, 1973b) in Y-PPase.…”