Modulation of Dimer Stability in Yeast Pyrophosphatase by Mutations at the Subunit Interface and Ligand Binding to the Active Site

Abstract

Yeast (Saccharomyces cerevisiae) pyrophosphatase (YPPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 M, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and phydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys 83 ), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg 2؉ cofactor, P i product, and the PP i analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k cat remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys 83 residue in the dimer.

A novel subfamily of monomeric inorganic pyrophosphatases in photosynthetic eukaryotes

“…reinhardtii)-sPPase I and Cr-sPPase II, which are located in the chloroplast and mitochondrion respectively. Although different genes of widely divergent phylo-geny encode them, both enzymes possess the conserved functional residues seen in all members of family I sPPases [2,5,6], but show changes in residues involved in oligomer stabilization [12]. Chloroplastic Cr-sPPase I is related to a eukaryotic sPPase, and we suggest that it replaced the ancestral cyanobacterial plastid sPPase at a very early stage of plastid evolution.…”

“…N-terminal extensions predicted to be chloroplastic transit peptides are indicated in grey boxes. All available chloroplastic sPPase sequences show substitutions (italicized and further indicated by arrowheads) of residues Arg 51 , Trp 52 and His 87 and lack of Trp 279 (yeast sPPase numbering, italics and bold) that are involved in yeast sPPase homodimer stabilization [12]. Peptides of purified Cr-sPPase I identified by MALDI-TOF fingerprint MS analysis are boxed in the protein sequence deduced from PPAI cDNA; they cover approx.…”

“…Chlorobium tepidum cells were kindly provided by Professor M. T. Madigan (Deparment of Microbiology, Southern Illinois University, Carbondale, IL, U.S.A.). The E. coli strain MC1061YPPA, which expresses the yeast YPPA gene instead of the native bacterial ppa gene [12], was kindly provided by Professor R. Lahti (Department of Biochemistry and Food Chemistry, University of Turku, Vatselankatu 2, FIN-20014 Turku, Finland).…”

Inorganic pyrophosphatases: structural diversity serving the function

“…In the case of yeast pyrophosphatase, the dimerization is favourable for substrate binding. 24 Dimeric forms of pyrophosphatases from eukaryotes and family II pyrophosphatases and hexameric forms of family I pyrophosphatases from prokaryotes (except for some organisms, which will be considered separately) ( Fig. 5) prevail in the nature.…”

“…24 Due to high crystallizability of PPases I, it was possible to obtain crystals of pyrophosphatases from S. cerevisiae and Thermococcus thioreducens (Tt-PPase) suitable for neutron diffraction studies. This method requires large crystals; the sizes of the crystals of Tt-PPase 33 were up to 5 mm 3 .…”