Probing Essential Water in Yeast Pyrophosphatase by Directed Mutagenesis and Fluoride Inhibition Measurements

Pekka Pohjanjoki,Igor P Fabrichniy,Vladimir N Kasho,Barry S Cooperman,Adrian Goldman,Alexander A Baykov,Reijo Lahti

doi:10.1074/jbc.m007360200

Abstract

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.

Highlights

Inorganic pyrophosphatase (EC 3.6.1.1; PPase)1 catalyzes reversible phosphoryl transfer from pyrophosphate (PPi) to water, a metabolically important reaction chemically similar to that catalyzed by numerous ATPases and GTPases
Mg2ϩ Binding—The effects of conservative mutations of Tyr93 and five amino acid ligands to Mg2ϩ (Fig. 1) on the binding of two activating metal ions in yeast pyrophosphatase (Y-PPase) active site were studied by equilibrium dialysis at pH 7.2 (Fig. 2)
The E48D variant, which exhibited the highest KM2 value (5.0 mM), is expected to be 50% saturated at the M2 site in the absence of substrate, but keeping in mind that substrate strengthens metal ion binding to the corresponding E-PPase E20D variant [22] and WT-E-PPase [23], at least, 90% saturation is expected in the presence of 200 ␮M Mg2PPi used in fluoride inhibition studies

Summary

EXPERIMENTAL PROCEDURES

The expression and purification of wild type Y-PPase and its active site variants from overproducing E. coli XL2blueb strain transformed with suitable plasmids were carried out as described by Heikinheimo et al [15]. The assay medium contained, except as noted, 0.32 mM (at pH 7.2) or 0.28 mM (at pH 8.5) total PPi (corresponding to 0.2 mM Mg2PPi complex), 5.5 mM MgCl2 (corresponding to 5 mM free Mg2ϩ), 0 –20 mM FϪ (added as NaF) and buffer. The assay medium used to measure fluoride effects on PPase in the presence of Pi contained 13.4 mM MgCl2 (5 mM free Mg2ϩ), 20 mM total Pi, 55 mM TES/KOH buffer, 11 mM KCl, and 33 ␮M EGTA. Values of the dissociation constants KM1 and KM2 for Mg2ϩ binding to two sites on Y-PPase were estimated by fitting equilibrium dialysis data to Equation 5, where n measures the number of Mg2ϩ ions bound per monomer [21]

RESULTS

41 Ϯ 7 62 Ϯ 5

DISCUSSION