The parent grant uses site-specific mutagenesis together with rate, equilibrium, and structural studies to fully characterize the active site structure and catalytic mechanism of S. cerevisiae pyrophosphatase (Y- PPase) and E. coli pyrophophatase (E-PPase). The present project also uses site-specific mutagenesis to study the role of quaternary structure in PPase catalysis and structural stability. Our studies will include: (1) structural character of wild-type and variant PPases by analytical ultracentrifugation and chemical cross-linking; (2) preparation of hybrid PPases having different mutations in different subunits; (3) determination of the effects of subunit dissociation and of hybrid PPase formation on functional properties (catalysis, metal-ion, Pi, PPi binding) of PPase; (4) calorimetric studies of the thermostabilities of wild-type and variant PPases. These studies will supplement those of the parent grant by detailing how changes in subunit:subunit contact modulate catalytic activity, thus allowing a more comprehensiVe analysis of structure:function relationships within PPases.
