AMP deaminase (AMPD) plays an integral role in fructose metabolism via its regulation by ATP, GTP and phosphate (Pi). The fructose catabolic pathway consumes ATP, producing ADP, which is further metabolized to AMP, triggering a cascade of reactions initiated by AMPD. This degradative pathway results in the final product uric acid, which is associated with metabolic acidosis, mitochondrial dysfunction, and gout. Understanding the regulation of the human liver AMPD isozyme (hAMPD2-2) under physiological conditions and under fructose consumption conditions will enable the design of targeted therapeutics to block the accumulation of uric acid. We report the first successful expression and purification from Escherichia coli of both the full-length and catalytic domains of hAMPD2-2. Steady-state kinetics confirmed allosteric activation by ATP of both the full-length and catalytic domains of hAMPD2-2 at physiological ATP concentrations (2-5 mM), suggesting that the allosteric ATP-binding site is located in the catalytic domain. Competitive inhibition by GTP of the ATP-activated enzyme, with Ki values of 74 and 101 M for the full-length and catalytic domains, respectively, was also consistent with this regulatory model. Pi, previously described in yeast AMPD as a competitive inhibitor, was shown to play a more nuanced role, that of enhancing inhibition of hAMPD2-2 when the enzyme is complexed to GTP, via competition at the ATP allosteric site. Pi binding thus further inhibits the pathway under normal physiological conditions, limiting production of cellular uric acid unless and until Pi and GTP levels are low.
Rebelo, A. M., Vuksanovic, N., Han, L., Tolan, D. R., Allen, K. N.
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