A tryptophan residue involved in the inhibition of plant vacuolar H+-ATPase by 2-hydroxy-5-nitrobenzyl bromide

Abstract

Treatment of the vacuolar H⁺ -ATPase from mung bean seedlings (Vigna radiata L.) with the tryptophan modifying agent 2-hydroxyl-5-nitrobenzyl bromide (HNBB), caused a progressive decline of the ATP hydrolysis activity and proton translocation in a time- and concentration-dependent manner. Dithiothreitol could not restore the inhibition of H⁺ -ATPase by HNBB, indicating possible involvement tryptophan, and not cysteine residues. Protection studies suggested that modified sites might not locate in the active domain. Kinetic analysis shows that V_max but not K_m of H⁺ -ATPase was changed by HNBB. The reaction order of inactivation by HNBB was calculated as 0.98, implying that at least one tryptophan was labelled. The steady-state dissociation constant (K_i) and the pesudo-first-order rate constant of inhibition (k₂) were determined as 1.61 mM and 0.22 min^-1, respectively. Furthermore, stoichiometry experiments indicated that 8 mol tryptophan/mol ATPase were modified, when the enzyme activity was completely inhibited. However, a Tsou analysis showed that only one out of these modified tryptophans was crucial to the enzymatic activity. In addition, modification of the vacuolar H⁺ -ATPase by HNBB led to a decrease in intrinsic fluorescence, suggesting a possible conformational change of the enzyme. Taken together, our data indicate that the tryptophan residue is indispensable to vacuolar H⁺ -ATPase and the modification of this residue may induce a significant conformational change, consequently resulting in the loss of enzymatic activity.