SITE-DIRECTED MUTAGENESIS OF VACUOLAR H-PYROPHOSPHATASE - NECESSITY OF CYS(634) FOR INHIBITION BY MALEIMIDES BUT NOT CATALYSIS()

A characteristic feature of the vacuolar H+-translocating inorganic py rophosphatase (V-PPase) of plant cells is its high sensitivity to irre versible inhibition by N-ethylmaleimide (NEM) and other sulfhydryl rea gents, Previous investigations in this laboratory have demonstrated th at the primary site for substrate-protectable covalent modification of the V-PPase by C-14-labeled NEM maps to a single M(r) 14,000 V8 prote ase fragment (V8(14K)) (Zhen, R.-G., Kim, E. J., and Rea, P. A. (1994) J. Biol. Chem. 269, 23342-23350). Here, we describe site-directed mut agenesis of the cDNA encoding the V-PPase hom Arabidopsis thaliana, it s heterologous expression in Saccharomyces cerevisiae and single subst itution of all 9 conserved Cys residues to either Ser or Ala. In all c ases, except one, Cys mutagenesis exerts little or no effect on either the catalytic activity or susceptibility of the enzyme to inhibition by NEM. By contrast, and in complete agreement with the results of pep tide mapping experiments, substitution of Cys(634), the sole conserved cysteine residue encompassed by V8(14K), with Ser or Ala generates en zyme that is insensitive to NEM but active in both PPi hydrolysis and PPi-dependent H+ translocation. The specific requirement for Cys(634) for inhibition by NEM and the dispensability of all of the conserved C ys residues, including Cys(634), for V-PPase function indicate that th e inhibitory action of maleimides reflects steric constraints imposed by the addition of a substituted alkyl group to the side chain of Cys( 634) rather than direct participation of this amino acid residue in ca talysis.