The Electrophilic and Leaving Group Phosphates in the Catalytic Mechanism of Yeast Pyrophosphatase

Abstract

Binding of pyrophosphate or two phosphate molecules to the pyrophosphatase (PPase) active site occurs at two subsites, P1 and P2. Mutations at P2 subsite residues (Y93F and K56R) caused a much greater decrease in phosphate binding affinity of yeast PPase in the presence of Mn(2+) or Co(2+) than mutations at P1 subsite residues (R78K and K193R). Phosphate binding was estimated in these experiments from the inhibition of ATP hydrolysis at a sub-K(m) concentration of ATP. Tight phosphate binding required four Mn(2+) ions/active site. These data identify P2 as the high affinity subsite and P1 as the low affinity subsite, the difference in the affinities being at least 250-fold. The time course of five "isotopomers" of phosphate that have from zero to four (18)O during [(18)O]P(i)-[(16)O]H(2)O oxygen exchange indicated that the phosphate containing added water is released after the leaving group phosphate during pyrophosphate hydrolysis. These findings provide support for the structure-based mechanism in which pyrophosphate hydrolysis involves water attack on the phosphorus atom located at the P2 subsite of PPase.

A Complete Structural Description of the Catalytic Cycle of Yeast Pyrophosphatase,

“…With the Mg 2+ -bound enzyme, P2 is released first, and in the structures presented here, the site is empty more often than not. This is consistent with earlier biochemical results (5) and provides support for the notion that the structures captured here represent typical states of the enzyme during catalysis. Consequently, we have used wt structures where possible to create a structural equivalent (Figure 9) of Scheme 2, which we have converted into a movie of catalysis (Supporting Information).…”

“…In addition, the P1 leaving group phosphate and the associated metal ion, M3, are bound, matching the contents expected for the F intermediate (Scheme 2) in the Mg 2+ -containing enzyme (5).…”

Reversible Inhibition of Escherichia coli Inorganic Pyrophosphatase by Fluoride: Trapped Catalytic Intermediates in Cryo-crystallographic Studies

“…A joint interpretation of the X-ray data and the results of solution kinetic studies suggest that P1 leaves first after the Mn-supported hydrolysis by E-PPase, while release of P2 is the rate-limiting step as it was shown for the yeast enzyme. 9 This structure discloses a principally new aspect of the problem of product release. It was proposed on a theoretical base that the rotation of the P2 phosphate is necessary for its release so that the O nu oxygen atom would be detached from the metal ions M1 and M2 and P2 would be brought to the P2 up position.…”

Inorganic pyrophosphatases: One substrate, three mechanisms

“…Family I PPases, first crystallised in 1952 [3], have been extensively-studied, especially those from Escherichia coli (EPPase) [13,21] and Saccharomyces cerevisiae (YPPase) [9,14,[22][23][24][25] well characterised. The work has included atomic resolution structures [25] as well as extensive structures of mutants that have led to a full structural description of the entire catalytic cycle [14].…”