{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:54:10Z","timestamp":1785430450687,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,10]]},"group-title":"Biochemistry","reference-count":37,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T00:00:00Z","timestamp":1781049600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"funder":[{"award":["U01 AA027997"],"award-info":[{"award-number":["U01 AA027997"]}],"id":[{"id":"https:\/\/ror.org\/02jzrsm59","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  AMP deaminase (AMPD) plays an integral role in fructose metabolism\n                  <jats:italic>via<\/jats:italic>\n                  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\n                  <jats:italic>Escherichia coli<\/jats:italic>\n                  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 K\n                  <jats:sub>i<\/jats:sub>\n                  values of 74 and 101 \u03bcM for the full-length and catalytic domains, respectively, was also consistent with this regulatory model. P\n                  <jats:sub>i<\/jats:sub>\n                  , 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,\n                  <jats:italic>via<\/jats:italic>\n                  competition at the ATP allosteric site. P\n                  <jats:sub>i<\/jats:sub>\n                  binding thus further inhibits the pathway under normal physiological conditions, limiting production of cellular uric acid unless and until P\n                  <jats:sub>i<\/jats:sub>\n                  and GTP levels are low.\n                <\/jats:p>","DOI":"10.64898\/2026.06.10.731346","type":"posted-content","created":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T03:10:15Z","timestamp":1781147415000},"source":"Crossref","is-referenced-by-count":0,"title":["Deciphering AMP deaminase-2 structure, activators and regulators underpinning cellular function in human fructose and nucleotide metabolism"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6691-6124","authenticated-orcid":false,"given":"Ashley M.","family":"Rebelo","sequence":"first","affiliation":[{"name":"Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston MA 02215"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2883-6312","authenticated-orcid":false,"given":"Nemanja","family":"Vuksanovic","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston MA 02215"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-8050-1882","authenticated-orcid":false,"given":"Lanlan","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston MA 02215"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0598-7241","authenticated-orcid":false,"given":"Dean R.","family":"Tolan","sequence":"additional","affiliation":[{"name":"Department of Biology, Boston University, 5 Cummington Mall, Boston MA 02215"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7296-0551","authenticated-orcid":false,"given":"Karen N.","family":"Allen","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston MA 02215"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026061218550668000_2026.06.10.731346v1.1","unstructured":"Average per capita consumption of sugar as of 2016, by country. Statista Inc., 2016. https:\/\/www.statista.com\/statistics\/535219\/global-sugar-per-capita-consumption-by-country\/ (accessed 2026."},{"key":"2026061218550668000_2026.06.10.731346v1.2","unstructured":"Added Sugars on the Nutrition Facts Label. Food and Drug Administration, 2026. https:\/\/www.fda.gov\/food\/nutrition-facts-label\/added-sugars-nutrition-facts-label (accessed 2026."},{"key":"2026061218550668000_2026.06.10.731346v1.3","doi-asserted-by":"publisher","DOI":"10.31883\/pjfns\/110735.The"},{"key":"2026061218550668000_2026.06.10.731346v1.4","doi-asserted-by":"publisher","DOI":"10.1002\/humu.24282"},{"key":"2026061218550668000_2026.06.10.731346v1.5","doi-asserted-by":"publisher","DOI":"10.1002\/humu.1380060303"},{"key":"2026061218550668000_2026.06.10.731346v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(88)90349-2"},{"key":"2026061218550668000_2026.06.10.731346v1.7","doi-asserted-by":"publisher","DOI":"10.1007\/s10545-010-9053-2"},{"key":"2026061218550668000_2026.06.10.731346v1.8","doi-asserted-by":"publisher","DOI":"10.3945\/an.112.002998"},{"key":"2026061218550668000_2026.06.10.731346v1.9","doi-asserted-by":"publisher","DOI":"10.1111\/j.1432-1033.1990.tb19225.x"},{"key":"2026061218550668000_2026.06.10.731346v1.10","doi-asserted-by":"publisher","DOI":"10.1111\/1440-1681.12162"},{"key":"2026061218550668000_2026.06.10.731346v1.11","doi-asserted-by":"publisher","DOI":"10.1016\/S0163-7258(00)00040-1"},{"key":"2026061218550668000_2026.06.10.731346v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-291X(03)00787-3"},{"issue":"31","key":"2026061218550668000_2026.06.10.731346v1.13","doi-asserted-by":"crossref","first-page":"22407","DOI":"10.1016\/S0021-9258(18)41686-9","article-title":"Molecular cloning of AMP deaminase isoform L. Sequence and bacterial expression of human AMPD2 cDNA","volume":"267","year":"1992","journal-title":"J Biol Chem"},{"key":"2026061218550668000_2026.06.10.731346v1.14","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.52.35118"},{"issue":"35","key":"2026061218550668000_2026.06.10.731346v1.15","first-page":"21422","article-title":"AMP deaminase from yeast. Role in AMP degradation, large scale purification, and properties of the native and proteolyzed enzyme","volume":"264","year":"1989","journal-title":"J Biol Chem"},{"key":"2026061218550668000_2026.06.10.731346v1.16","doi-asserted-by":"publisher","DOI":"10.1042\/bj1620601"},{"issue":"8","key":"2026061218550668000_2026.06.10.731346v1.17","doi-asserted-by":"crossref","first-page":"4420","DOI":"10.1016\/S0021-9258(19)39581-X","article-title":"Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism","volume":"265","year":"1990","journal-title":"J Biol Chem"},{"key":"2026061218550668000_2026.06.10.731346v1.18","doi-asserted-by":"publisher","DOI":"10.1107\/S2053230X15000187"},{"key":"2026061218550668000_2026.06.10.731346v1.19","unstructured":"Segel, I. H. Enzyme kinetics, behavior and analysis of rapid equilibrium and steady-state enzyme systems; Wiley-Interscience, 1975."},{"key":"2026061218550668000_2026.06.10.731346v1.20","doi-asserted-by":"publisher","DOI":"10.1007\/BF00928361"},{"key":"2026061218550668000_2026.06.10.731346v1.21","doi-asserted-by":"publisher","DOI":"10.1002\/mrm.26635"},{"issue":"1","key":"2026061218550668000_2026.06.10.731346v1.22","first-page":"81","article-title":"Human liver AMP-deaminase \u2013 oligomeric forms of the enzyme","volume":"241","year":"2002","journal-title":"Molecular & Cellular Biochemistry"},{"issue":"6","key":"2026061218550668000_2026.06.10.731346v1.23","doi-asserted-by":"crossref","first-page":"4549","DOI":"10.1016\/S0021-9258(18)53645-0","article-title":"Yeast AMP deaminase. Catalytic activity in Schizosaccharomyces pombe and chromosomal location in Saccharomyces cerevisiae","volume":"268","year":"1993","journal-title":"J Biol Chem"},{"key":"2026061218550668000_2026.06.10.731346v1.24","doi-asserted-by":"publisher","DOI":"10.1016\/0005-2744(79)90210-9"},{"key":"2026061218550668000_2026.06.10.731346v1.25","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2016.09.009"},{"key":"2026061218550668000_2026.06.10.731346v1.26","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.93.12.5720"},{"key":"2026061218550668000_2026.06.10.731346v1.27","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E11-08-0699"},{"key":"2026061218550668000_2026.06.10.731346v1.28","doi-asserted-by":"publisher","DOI":"10.3389\/fnut.2022.1045805"},{"issue":"1","key":"2026061218550668000_2026.06.10.731346v1.29","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1007\/BF00683216","article-title":"Purification and Properties of Trout Gill AMP Deaminase","volume":"154","year":"1984","journal-title":"Journal of Comparative Physiology B-Biochemical Systemic and Environmental Physiology"},{"key":"2026061218550668000_2026.06.10.731346v1.30","doi-asserted-by":"publisher","DOI":"10.1002\/hep.25741"},{"key":"2026061218550668000_2026.06.10.731346v1.31","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.399899"},{"issue":"6","key":"2026061218550668000_2026.06.10.731346v1.32","doi-asserted-by":"crossref","first-page":"3668","DOI":"10.1016\/S0021-9258(18)32717-0","article-title":"Platelet AMP deaminase. Regulation by Mg-ATP2- and inorganic phosphate and inhibition by the transition state analog coformycin","volume":"258","year":"1983","journal-title":"J Biol Chem"},{"key":"2026061218550668000_2026.06.10.731346v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/s0021-9258(19)81037-2"},{"key":"2026061218550668000_2026.06.10.731346v1.34","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2024.102974"},{"key":"2026061218550668000_2026.06.10.731346v1.35","doi-asserted-by":"publisher","DOI":"10.1096\/fj.13-243634"},{"key":"2026061218550668000_2026.06.10.731346v1.36","doi-asserted-by":"publisher","DOI":"10.3803\/EnM.2016.31.4.519"},{"key":"2026061218550668000_2026.06.10.731346v1.37","doi-asserted-by":"publisher","DOI":"10.1126\/science.1247363"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.06.10.731346","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,13]],"date-time":"2026-06-13T01:55:15Z","timestamp":1781315715000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.06.10.731346"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,10]]},"references-count":37,"URL":"https:\/\/doi.org\/10.64898\/2026.06.10.731346","relation":{},"subject":[],"published":{"date-parts":[[2026,6,10]]},"subtype":"preprint"}}