Structural Studies of the Vacuolar H+-Pyrophosphatase: Sequence Analysis and Identification of the Residues Modified by Fluorescent Cyclohexylcarbodiimide and Maleimide

Abstract

We determined the amino acid residues of the H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) of pumpkin which are covalently labeled by two fluorescent labeling reagents; N-cyclohexyl-N'-[4-(dimethyl amino)-alpha-naphthyl] carbodiimide (NCD) and N-pyrenylmaleimide (NPM). NCD and NPM are fluorescent analogues of N,N-dicycrohexylcarbodiimide and N-ethylmaleimide, respectively, and inactivate H(+)-PPase activity. Excess Mg2+ protected the H(+)-PPase from the inactivation by these reagents. Furthermore, we identified the cDNA clone encoding the pumpkin H(+)-PPase in order to determine the position of labeled residues. The nucleotide sequence of the cDNA clone contains a 2,304 bp open reading frame encoding a polypeptide with 768 amino acids. Chemical sequence analysis of fluorescent peptide fragments revealed that Glu749 located in the C-terminal putative transmembrane alpha-helix was a NCD-labeled residue, and Cys632 was a NPM-labeled residue located in a putative cytosolic domain. The amino acid sequence of the region that includes Glu749 is highly conserved in H(+)-PPases from other plants and it also shows some sequence similarity with the region of the carbodiimide-reactive Glu (or Asp) of F0F1-ATPase c-subunit. The reactive glutamic acids in these proteins are located at the last C-terminal transmembrane alpha-helix. We also found that the H(+)-PPase shows significant amino acid sequence similarity to Kdp-ATPase A chain of E. coli. This similarity between the two different proteins suggest that they have evolved from a common ancestor and may utilize a common basic mechanism for ion transport.

Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

“…There is some similarity of KdpA to the sequence of some plant pyrophosphatases that couple H ϩ transport to synthesis or hydrolysis of pyrophosphate (29). However, most of the regions of similarity are outside the clusters, and only a few residues in clusters are included.…”

H+‐proton‐pumping inorganic pyrophosphatase: a tightly membrane‐bound family

“…Nine amino acid sequences of plant V‐PPases have been solved and shown to form a distinct protein family [13–21]. Two are from green algae and have been published very recently [20,21].…”

Mechanisms of proton translocation in Methanosarcina mazei Gö1

“…This sequence encompasses a Glu residue also implicated in coupling PPi hydrolysis and H + -translocation (30). iii) A carbodiimide-reactive Glu residue has been identified within the C-terminal part of the enzyme from pumpkin (33). It was proposed that the DCCD-sensitive residue is near by the Mg 2+ -binding site.…”