Effect of pH on Tobacco Anionic Peroxidase Stability and Its Interaction with Hydrogen Peroxide

I. G. Gazaryan^1*, I. V. Ouporov², T. A. Chubar¹, V. A. Fechina³, E. A. Mareeva¹, and L. M. Lagrimini⁴

¹Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-5417

²Department of Chemistry, University of North Dakota, Grand Forks, ND 58202, USA

³Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia

⁴Department of Horticulture and Crop Science, Ohio State University, Columbus OH 43210-1096, USA

^* To whom correspondence should be addressed.

Received October 23, 1997; Revision received November 20, 1997

---

The effect of extremely acidic pH on the stability of tobacco peroxidase and lignin peroxidase holoenzymes has been studied. Stabilization of tobacco peroxidase holoenzyme in the presence of calcium cations at pH < 2 and stabilization of lignin peroxidase at pH > 2 in the presence of veratryl alcohol have been shown. The dependence of the reaction rate constant for hydrogen peroxide interaction with tobacco peroxidase on pH suggests that the reaction rate is under control of a group with pK of 2.5. A tobacco peroxidase model structure has been created by means of homology modeling on the basis of the tobacco peroxidase sequence and the coordinates of peanut peroxidase crystal structure. The model structure demonstrates the presence of the negatively charged Glu-141 at the entrance to the active site and its electrostatic repulsion from heme propionates and triad of Asp-76, -79, and -80 residues. The results on tobacco holoperoxidase stabilization at pH 1.8 in the presence of calcium cations and drop in reaction rate constant for the enzyme interaction with hydrogen peroxide are explained by a hypothetical formation of ionic bonds between Glu-141 and the triad of aspartic acid residues via calcium cation lowering the accessibility of the active site and stabilizing the holoenzyme.

---

KEY WORDS: calcium, magnesium, veratryl alcohol, model structure, lignin peroxidase, holoenzyme, stability

---