
TY  - JOUR
A1  - Kuo, Soong Yu 
A1  - Lin, May Whei 
A1  - Jiang, Shih Sheng 
A1  - Hung, Shu Hsien 
A1  - Tzeng, Chi Meng 
A1  - Pan, Rong Long 
T1  - A tryptophan residue involved in the inhibition of plant vacuolar H<emph type="7">+</emph>-ATPase by 2-hydroxy-5-nitrobenzyl bromide
JF  - Functional Plant Biology
JO  - Functional Plant Biol.
Y1  - 1998
VL  - 25
IS  - 6
SP  - 679
EP  - 687
KW  - vacuole, tonoplast, H+ -ATPase, chemical modification, tryptophan.
UR  - https://doi.org/10.1071/PP98008 
N2  - 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 Vmax but not Km 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 (Ki) 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. 
ER  -

