Involvement of tyrosine residue in the inhibition of plant vacuolar H +-pyrophosphatase by tetranitromethane

• Published in: Biochimica et biophysica acta 1996-05, Vol.1294 (1), p.89-97
• Main Authors: Yang, Su Jing, Jiang, Shih Sheng, Tzeng, Chi Meng, Kuo, Soong Yu, Hung, Shu Hsien, Pan, Rong Long
• Format: Article
• Language: English
• Subjects: Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - pharmacology; Fabaceae - enzymology; Inorganic Pyrophosphatase; Kinetics; Plants, Medicinal; Pyrophosphatase; pyrophosphatases; Pyrophosphatases - antagonists & inhibitors; Pyrophosphatases - isolation & purification; Pyrophosphatases - metabolism; Spectrophotometry; Tetranitromethane; Tetranitromethane - pharmacology; Tonoplast; Tyrosine - metabolism; Vacuoles - enzymology; Vigna radiata
• ISSN: 0167-4838; 0006-3002; 1879-2588; 1878-2434
• DOI: 10.1016/0167-4838(96)00005-2

Plant vacuolar vesicles contain a novel H +-translocating pyrophosphatase (H +-PPase, EC 3.6.1.1). Modification of tonoplast vesicles and purified vacuolar H +-PPase from etiolated mung bean seedlings with tetranitromethane (TNM) resulted in a progressive decline in H +-translocating pyrophosphatase activity. The half-maximal inhibition was brought about by 0.6, 1.0, and 0.8 mM TNM for purified and membrane-bound H +-PPases, and its associated proton translocation, respectively. The maximal inhibition of vacuolar H +-PPase by TNM occurred at a pH value above 8. Loss of activity of purified H +-pyrophosphatase followed pseudo-first order rate kinetics, yielding a first-order rate constant ( k 2) of 0.039 s −1 and a steady-state dissociation constant of inactivation ( K i) of 0.02 mM. Covalent modification of vacuolar H +-PPase by TNM increased K m value of the enzyme for its substrate without a significant effect on V max. Double logarithmic plots of the pseudo-first order rate constant ( k obs) versus TNM concentration exhibited a slope of 0.88, suggesting that at least one tyrosine residue was involved in the inactivation of H +-PPase enzymatic activity. Further spectrophotometric measurements of the nitrated H +-pyrophosphatase indicated that TNM could modify approximately two tyrosine residues/subunit of the enzyme. However, Tsou's analysis revealed that only one of those modified tyrosine residues directly participated in the inhibition of enzymatic activity of vacuolar H +-PPase. The physiological substrate, i.e., dimagnesium pyrophosphate, provided substantial protection against inactivation by TNM. Moreover, NEM pretreatment of the enzyme decreased the number of subsequent nitration of vacuolar H +-PPase. Taken together, we suggest that vacuolar H +-pyrophosphatase contains a substrate-protectable tyrosine residue conferring to the inhibition of its activity and this tyrosine residue may be located in a domain sensitive to the modification of Cys-634 by NEM.