{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:28:31Z","timestamp":1785414511043,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,4]]},"group-title":"Biochemistry","reference-count":68,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T00:00:00Z","timestamp":1780531200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["GM121849"],"award-info":[{"award-number":["GM121849"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["GM127144"],"award-info":[{"award-number":["GM127144"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Chemical potential is coupled to cellular processes by the flow of metabolites through catalytic networks known collectively as metabolism. Here we describe an extensive new class of energy-coupling catalysts that act to interconnect metabolic network pathways and their potentials. Members of the class are defined by a common mechanism \u2014 half-site reactivity. The well-established sequential subunit turnover of half-site enzymes suggests that the potentials of reactions occurring at the separate subunits are coupled to one another. Here this hypothesis is tested and validated using promiscuous half-site enzymes from two catalytically distinct enzyme families, each with broad metabolic penetrance. Fundamental catalytic parameters (V\n                  <jats:sub>max<\/jats:sub>\n                  and K\n                  <jats:sub>m<\/jats:sub>\n                  ) and reaction endpoints are predicted and shown to change dramatically when reaction potentials are coupled \u2014 for example, the catalytic efficiency (V\n                  <jats:sub>max<\/jats:sub>\n                  \/K\n                  <jats:sub>m<\/jats:sub>\n                  ) and endpoint of the retinol oxidation reaction (the rate-limiting step in vitamin A synthesis) are shown to increase 900- and 3,400-fold, respectively, when the reaction is coupled to the more favorable oxidation of ethanol. For the first time it is clear that metabolism has the flexibility to react to changes in the metabolic state of the cell by redistributing chemical potential among the many metabolic pathways interconnected by half-site enzymes.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance<\/jats:title>\n                  <jats:p>\n                    The findings herein reveal the existence of an extensive, catalytically diverse network of enzymes that distributes chemical potential within and across the pathways of small-molecule metabolism. Members of the network are identified on the basis of a shared mechanistic trait \u2014 half-site reactivity. These energy-coupling catalysts allow reactions to proceed orders of magnitude further and more efficiently than their intrinsic potentials allow by coupling them to more favorable reactions. The work offers a\n                    <jats:italic>raison d\u2019etre<\/jats:italic>\n                    for the half-site mechanism and powerful new strategies for\n                    <jats:italic>de novo<\/jats:italic>\n                    metabolic pathway design.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.01.729314","type":"posted-content","created":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T19:55:14Z","timestamp":1780602914000},"source":"Crossref","is-referenced-by-count":0,"title":["On the Distribution of Free-Energy in Metabolism"],"prefix":"10.64898","author":[{"given":"Ian","family":"Cook","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, New York 10461-1926"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1541-5204","authenticated-orcid":false,"given":"Thomas S.","family":"Leyh","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, New York 10461-1926"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026060713100458000_2026.06.01.729314v1.1","doi-asserted-by":"publisher","DOI":"10.1007\/3-540-36466-8_5"},{"key":"2026060713100458000_2026.06.01.729314v1.2","doi-asserted-by":"publisher","DOI":"10.1007\/s10540-005-2739-2"},{"key":"2026060713100458000_2026.06.01.729314v1.3","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.X200001200"},{"key":"2026060713100458000_2026.06.01.729314v1.4","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R117.001324"},{"key":"2026060713100458000_2026.06.01.729314v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nrm2774"},{"key":"2026060713100458000_2026.06.01.729314v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/386299a0"},{"key":"2026060713100458000_2026.06.01.729314v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/nrm3667"},{"key":"2026060713100458000_2026.06.01.729314v1.8","doi-asserted-by":"publisher","DOI":"10.1097\/00003246-198902000-00019"},{"key":"2026060713100458000_2026.06.01.729314v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2017.10.005"},{"key":"2026060713100458000_2026.06.01.729314v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.csbj.2019.07.020"},{"key":"2026060713100458000_2026.06.01.729314v1.11","doi-asserted-by":"publisher","DOI":"10.1038\/525018a"},{"key":"2026060713100458000_2026.06.01.729314v1.12","doi-asserted-by":"publisher","DOI":"10.1039\/C7CS00245A"},{"key":"2026060713100458000_2026.06.01.729314v1.13","doi-asserted-by":"crossref","first-page":"3794","DOI":"10.1016\/S0021-9258(18)32735-2","article-title":"Malate thiokinase. 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