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In this study, we investigated human phosphofructokinase\u20101 platelet isoform (PFKP) in different crowding conditions. Temperature\u2010dependent studies allowed us to i) measure the robustness of enzymatic activity in different environments, ii) decipher the thermodynamic contributions that govern the associated crowding effects. Folding stability and assembly of PFKP changed under crowding as expected by excluded\u2010volume considerations; however, crowder\u2010specific interactions with substrates and the active site region become important when considering catalytic activity. Enzyme kinetics showed Arrhenius\u2010like behavior at low temperatures but strong deviations at elevated temperatures. We found that the temperature robustness in the non\u2010Arrhenius regime is increased by glucose and dextran. Using Macromolecular Rate Theory, we could attribute this effect to the crowders restricting conformational fluctuations leading to the transition state. We found that a delicate balance of enthalpic and entropic crowding effects determines enzymatic activity and temperature robustness in different environments. Deciphering the respective contributions for different enzymes and cell\u2010like conditions will be important to understand the interplay between the local cellular environment and enzyme activity.<\/jats:p>","DOI":"10.1002\/anie.3134204","type":"journal-article","created":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T04:12:22Z","timestamp":1791519142000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Soft Interactions, Rather Than Crowder Size, Determine Deviations From Arrhenius\u2010Like Kinetics of Phosphofructokinase\u20101"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4300-6923","authenticated-orcid":false,"given":"Harshita","family":"Rastogi","sequence":"first","affiliation":[{"name":"Lehrstuhl f\u00fcr Biophysikalische Chemie Ruhr\u2010Universit\u00e4t Bochum  Bochum Germany"},{"name":"Research Center Chemical Sciences and Sustainability Research Alliance Ruhr Bochum Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-0661-2584","authenticated-orcid":false,"given":"Madhav","family":"Samanta","sequence":"additional","affiliation":[{"name":"Heyrovsky Institute Czech Academy of Sciences  Prague Czech Republic"},{"name":"Faculty of Mathematics and Physics Charles University  Prague Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6497-9033","authenticated-orcid":false,"given":"Truong\u2010An","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Heyrovsky Institute Czech Academy of Sciences  Prague Czech Republic"},{"name":"Faculty of Science Charles University  Prague Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0563-0947","authenticated-orcid":false,"given":"Mehrnoosh Khodam","family":"Hazrati","sequence":"additional","affiliation":[{"name":"Heyrovsky Institute Czech Academy of Sciences  Prague Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5299-2852","authenticated-orcid":false,"given":"Bradley A.","family":"Webb","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Medicine West Virginia University  Morgantown West Virginia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4167-4891","authenticated-orcid":false,"given":"Eva","family":"Pluha\u0159ov\u00e1","sequence":"additional","affiliation":[{"name":"Heyrovsky Institute Czech Academy of Sciences  Prague Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5824-4476","authenticated-orcid":false,"given":"Stepan","family":"Timr","sequence":"additional","affiliation":[{"name":"Heyrovsky Institute Czech Academy of Sciences  Prague Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9309-1279","authenticated-orcid":false,"given":"Simon","family":"Ebbinghaus","sequence":"additional","affiliation":[{"name":"Lehrstuhl f\u00fcr Biophysikalische Chemie Ruhr\u2010Universit\u00e4t Bochum  Bochum Germany"},{"name":"Research Center Chemical Sciences and Sustainability Research Alliance Ruhr Bochum Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,8]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.56.070187.000513"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1152241"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaz6465"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2017.06.082"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2019.06.013"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00007a019"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2023.10.033"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.104.050351"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12551\u2010013\u20100108\u20100"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R100005200"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi5011255"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi100727y"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/jz5002715"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev\u2010biophys\u2010091321\u2010071829"},{"key":"e_1_2_8_16_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.bbrep.2021.100956","article-title":"Macromolecular Crowding Effects on the Kinetics of Opposing Reactions Catalyzed by Alcohol Dehydrogenase","volume":"26","author":"Wilcox X. 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