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.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
5R03TW000407-03
Application #
2292002
Study Section
Special Emphasis Panel (SRC)
Project Start
1994-09-01
Project End
1998-08-31
Budget Start
1996-09-01
Budget End
1998-08-31
Support Year
3
Fiscal Year
1996
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104