Characterization of an archaeal inorganic pyrophosphatase from Sulfolobus islandicus using a [31P]-NMR-based assay

Abstract

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [³¹P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [³¹P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg²⁺ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The V_max value was 850 μmol/min/mg and the K_m for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC₅₀ of 2.26 mM, compared to a IC₅₀ of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [³¹P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes.

Keywords: Assay; Enzyme; Phosphate; Pyrophosphatase; Sulfolobus islandicus; [(31)P]-NMR.