title: Involvement of tyrosine residue in the inhibition of plant vacuolar H(+)-pyrophosphatase by tetranitromethane.
abstract: 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 (k2) of 0.039 s(-1) and a steady-state dissociation constant of inactivation (Ki) of 0.02 mM. Covalent modification of vacuolar H(+)-PPase by TNM increased Km value of the enzyme for its substrate without a significant effect on Vmax. Double logarithmic plots of the pseudo-first order rate constant (kobs) 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.
authors.affiliation: Institute of Radiation Biology, College of Nuclear Science, National Tsing Hua University, Taiwan, Republic of China.
authors.forename: S J
authors.initials: SJ
authors.lastname: Yang
authors.forename: S S
authors.initials: SS
authors.lastname: Jiang
authors.forename: C M
authors.initials: CM
authors.lastname: Tzeng
authors.forename: S Y
authors.initials: SY
authors.lastname: Kuo
authors.forename: S H
authors.initials: SH
authors.lastname: Hung
authors.forename: R L
authors.initials: RL
authors.lastname: Pan
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007887:Fabaceae / Q000201:enzymology*; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D010946:Plants, Medicinal; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013774:Tetranitromethane / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D014443:Tyrosine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D013774:Tetranitromethane