A1 Journal article – refereed
Modulation of dimer stability in yeast pyrophosphatase by mutations at the subunit interface and ligand binding to the active site

List of Authors: Salminen A, Parfenyev AN, Salli K, Efimova IS, Magretova NN, Goldman A, Baykov AA, Lahti R
Publication year: 2002
Journal: Journal of Biological Chemistry
Journal acronym: J BIOL CHEM
Volume number: 277
Issue number: 18
Number of pages: 7
ISSN: 0021-9258

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) 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 muM, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH greater than or equal to 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 p-hydroxymercurobenzosulfonate 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 (Mg2+ cofactor, P-i product, and the PPi 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.

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