Halonen Pasi, Baykov Alexander A, Goldman Adrian, Lahti Reijo, Cooperman Barry S
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
Biochemistry. 2002 Oct 8;41(40):12025-31. doi: 10.1021/bi026018z.
Soluble inorganic pyrophosphatase (PPase), which converts inorganic pyrophosphate (PP(i)) into usable phosphate, is almost universally present as a central enzyme of phosphorus metabolism and uses divalent metal ion as a necessary cofactor. PPase from Saccharomyces cerevisiae (Y-PPase) is the best studied with respect to both structure and mechanism. Here we report the first combined use of stopped flow and quenched flow techniques to study the PPase reaction in both the forward (PP(i) hydrolysis) and back (PP(i) synthesis) directions. The results of these studies permit direct comparison of different divalent metal-ion effects (Mg(2+), Mn(2+), Co(2+)) on microscopic rate constants at pH 7.0. For the Mn-enzyme, on which all of the high-resolution X-ray studies have been conducted, they demonstrate that the rate-determining step changes as a function of pH, from hydrolysis of enzyme-bound PP(i) at low pH to release of the more tightly bound P(i) at high pH. They also provide evidence for two kinetically important forms of the product complex EM(4)(P(i))(2), supporting an earlier suggestion based on crystallographic evidence, and allow informed speculation as to the identities of acidic and basic groups essential for optimal PPase catalytic activity.
