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J. Biol. Chem., Vol. 277, Issue 18, 15465-15471, May 3, 2002

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

Anu Salminen^§, Alexey N. Parfenyev^§¶, Krista Salli, Irina S. Efimova^¶, Natalia N. Magretova^¶, Adrian Goldman, Alexander A. Baykov^¶**, and Reijo Lahti

From the  Department of Biochemistry, University of Turku, FIN-20500 Turku, Finland, ^¶ A. N.Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia, and  Institute of Biotechnology, University of Helsinki, P. O. Box 56, FIN-00014 Helsinki, Finland

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 µM, 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 p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys⁸³), 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 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 Cys⁸³ residue in the dimer.

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