W0113

Catalytic Mechanism of Inorganic Pyrophosphatase. Vesa Tuominen^1,2, Pirkko Heikinheimo¹, Reijo Lahti², Barry S. Cooperman³ & Adrian Goldman^1,2, ¹Turku Centre for Biotechnology, PO. Box 123, FIN-20521 Turku, Finland, ²Department of Biochemistry, University of Turku, FIN-20014 University of Turku, Finland, ³Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104 U.S.A.

Inorganic pyrophosphatase (PPase) catalyses the hydrolysis of metal coordinated inorganic pyrophosphate. It is thus an essential enzyme for cell growth and is present in all living organisms.

Our product complex crystals of Saccharomyces cerevisiae PPase diffract to 1.1 Å (at 1.5 Å resolution, R-factor 19.2%) and by seeding they can be grown as large as 2 mM on a side, suitable for neutron diffraction. These studies will help to solve the last unclear details of the reaction mechanism. We have also solved the structure of two yeast PPase active-site variant product complexes, R78K and D117E, at resolution of 1.85 and 2.15 Å and R-factors of 19.5% and 18.3 %.¹

These and earlier mutagenesis, mechanistic and structural studies have shown that the attacking nucleophile is most likely a water molecule (Wat1) coordinated to two metals in the enzyme active site, in a manner chemically similar that of the polymerases.^2,3 These new structures also support other details of our model for catalysis.

1. Tuominen, V., Heikinheimo, P., Kajander, T., Torkkel, T., Käpylä, J., Hyytiä, T., Lahti R., Baykov, A., Cooperman, B. S., & Goldman A. (Manuscript)

2. Heikinheimo, P., Lehtonen J., Baykov, A., Lahti, R., Cooperman, B. S. & Goldman, A. (1996) Structure, 4, 1491.

3. Pohjanjoki, P., Lahti R., Goldman, A. & Cooperman B. S. (1998) Biochemistry, in Press.