Biochemistry Biochemistry最新文献_第4页

Enzyme Catalysis: The Effective Molarity for Fragments of Enzyme-Bound Whole Substrates or Active-Site Amino Acid Side Chains 酶催化：酶结合的整个底物或活性位点氨基酸侧链片段的有效摩尔浓度

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-10-06 Epub Date: 2026-09-18 DOI: 10.1021/acs.biochem.6c00503

John P. Richard

{"title":"Enzyme Catalysis: The Effective Molarity for Fragments of Enzyme-Bound Whole Substrates or Active-Site Amino Acid Side Chains","authors":"John P. Richard","doi":"10.1021/acs.biochem.6c00503","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00503","url":null,"abstract":"Truncating an accessory fragment from the substrate orsubstitutingan active-site side chain in the enzyme catalyst generally resultsin a decrease in kcat/Km for the catalyzed reaction, and the activity lost maysometimes be recovered in third-order reactions of the substrate orenzyme fragments. The effective molarity (EM) for a fragment truncatedfrom the substrate or for a substituted active-site side chain atthe transition state for the whole enzyme-catalyzed reaction is equalto kcat/Km (M–1 s–1) for the wild-typeenzyme-catalyzed reaction divided by the rate constant (M–2 s–1) for the third-order enzyme-catalyzed reactionof the substrate or enzyme pieces. These EMs are the predicted concentrationof the deleted substrate or enzyme piece in the third-order reactionrequired to give the reaction velocity observed for the wild-typeenzyme-catalyzed reaction. The EMs determined for a variety of enzymaticreactions range from 4 to 109 M. These variations are interpretedusing a model that assumes an ≈6 kcal/mol advantage for thereaction of whole species that arises from the larger entropic barrierto the higher-order reactions of enzyme or substrate fragments. Interactionsthat specifically stabilize or destabilize the transition states forenzyme-catalyzed reactions of the fragments also affect the EM. LargeEMs are observed when fragment binding to the enzyme is impaired,and small EMs result when the fragment complex is stabilized by interactionsbetween second-shell active-site side chain(s). The reported EM valuesare largely entropically controlled, consistent with a high degreeof structural organization at the active sites for the wild-type andvariant enzymes.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 19","pages":"3044–3056"},"PeriodicalIF":2.7,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149124786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cellulose-Binding Module-Mediated Immobilization of l-Arabinose Isomerase for Reusable d-Tagatose Production 纤维素结合模块介导的l-阿拉伯糖异构酶固定化用于可重复使用的d-塔格糖生产

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-10-06 Epub Date: 2026-09-10 DOI: 10.1021/acs.biochem.6c00239

Amol D. Pagar, Sonia Su, Zhixin Wang, Julie M. Goddard

{"title":"Cellulose-Binding Module-Mediated Immobilization of l-Arabinose Isomerase for Reusable d-Tagatose Production","authors":"Amol D. Pagar, Sonia Su, Zhixin Wang, Julie M. Goddard","doi":"10.1021/acs.biochem.6c00239","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00239","url":null,"abstract":"The development of stable and recyclable biocatalystsis vitalfor sustainable bioprocessing. In this study, we demonstrated fusionof l-arabinose isomerase from Thermotoga neapolitana (TnAI) with a cellulose-binding module (CBM) to facilitate direct,noncovalent immobilization on cellulose fibers for the biocatalytictransformation of d-galactose to d-tagatose. Theimpact of His-tag positioning on TnAI activity and quaternary structurewas initially assessed, revealing that the C-terminal tag caused enzymeaggregation and loss of activity. In contrast, the N-terminal His-tagpreserved the hexameric configuration and catalytic efficiency. ThreeCBM-TnAI fusion variants containing flexible linkers (L1–L3)were subsequently generated, expressed, and purified. All CBM-fusedvariants retained solubility and catalytic activity comparable tothe native enzyme, confirming that the N-terminal CBM fusion doesnot disrupt active-site conformation. Immobilization studies on microcrystallinecellulose showed high binding efficiencies (>80%) and substantialretention of catalytic activity, with CBM_L2_TnAI exhibiting the highestcellulose-binding efficiency (>90%) and immobilized activity (∼87%).Consistent with its superior catalytic performance and immobilizationcharacteristics, CBM_L2_TnAI was selected for reusability studiesand retained approximately 40% of its initial activity after fivereaction cycles, demonstrating good operational stability. This CBM-mediatedimmobilization strategy eliminates the need for chemical cross-linkers,providing a simple, cost-effective, and sustainable platform for producingreusable enzyme systems. The cellulose-protein hybrid catalyst developedherein offers a versatile framework for continuous or batch biotransformationof low-value waste carbohydrates into value-added rare sugars.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 19","pages":"3057–3065"},"PeriodicalIF":2.7,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149093530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Conserved Hinge Motif Modulates Conformational Mobility of a Heme Trafficking Protein from Fusobacterium nucleatum 保守的铰链基序调节核梭杆菌血红素运输蛋白的构象迁移

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-10-06 Epub Date: 2026-09-23 DOI: 10.1021/acs.biochem.6c00314

Stella I. Widjaja, M. Hossein Khalilian, Alexandra K. McGregor, Kirsten R. Wolthers

{"title":"Conserved Hinge Motif Modulates Conformational Mobility of a Heme Trafficking Protein from Fusobacterium nucleatum","authors":"Stella I. Widjaja, M. Hossein Khalilian, Alexandra K. McGregor, Kirsten R. Wolthers","doi":"10.1021/acs.biochem.6c00314","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00314","url":null,"abstract":"Fusobacterium nucleatum encodesHmuF, which has been shown in vitro to bind tightlyto heme, traffic the cofactor to anaerobilin synthase, and catalyzethe four-electron reduction of anaerobilin. The structure of the heme-boundHmuF paralog, FldH, reveals an α-helical cap appended to theflavodoxin domain. Molecular dynamics simulations of FldH withoutheme reveal that the α-helical cap pivots away from the flavodoxindomain to form an open complex, but then reforms the closed complex,which remains closed for the duration of the MD simulation. Transitionto the open conformation disrupts noncovalent interactions formedby three conserved residues (cis-Pro151, His150,and Asp155) that form a hinge bisecting the cap and flavodoxin domain. 19F NMR was used to probe the dynamics of the cap and the roleof the hinge motif. 1D NMR reveals that a single 19F-Tyrprobe in the α-helical cap has two resonances that slowly exchange(τ = 1.5 ms). A H150A and D155N substitution caused coalescenceof these two peaks, indicating faster exchange or stabilization ofone conformer. A P151G substitution not only led to a shift in the 19F-Tyr128 peak(s), but also to the chemical shifts of the 19F-Tyr residues located in the flavodoxin fold, indicatinga greater perturbation in protein structure. Despite the variantsexhibiting different 19F-NMR spectra, all engage in hemetrafficking and anaerobilin reduction. Together, these results indicatethat while the conserved hinge fine-tunes the dynamics of the α-helicalcap, this motif is not a strict prerequisite for HmuF/FldH function.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 19","pages":"3066–3079"},"PeriodicalIF":2.7,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149093652","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

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Cryptic RNA Binding Sites Are Energetically Accessible and Chemically Addressable 隐RNA结合位点是能量可及和化学可寻址的

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-09-15 Epub Date: 2026-08-29 DOI: 10.1021/acs.biochem.6c00540

Lukasz T. Olenginski, Robert T. Batey

{"title":"Cryptic RNA Binding Sites Are Energetically Accessible and Chemically Addressable","authors":"Lukasz T. Olenginski, Robert T. Batey","doi":"10.1021/acs.biochem.6c00540","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00540","url":null,"abstract":"Cryptic binding sites generated by local conformationaldynamicshave become an important concept in protein-targeted ligand discovery,yet their energetic accessibility and relevance to RNA recognitionremain less well-understood. Here, we use the env8 cobalamin (Cbl) riboswitch as a model system to investigate theenergetic consequences of cryptic-site formation through base displacement.Structural analysis revealed that binding of β-axial-substitutedCbl derivatives displaces a conserved adenosine (A20) from the RNAcore, exposing a previously hidden binding site occupied by the β-axialsubstituent. Using selective abasic substitution, we quantified theenergetic contributions of A20 within the native RNA core and itsdisplacement. Isothermal titration calorimetry and fluorescence measurementsrevealed that cryptic-site formation incurs a modest energetic penaltyof ∼1.4 kcal mol–1. Guided by this experimentallyderived framework, computational conformational sampling recapitulatedcryptic-site formation through A20 displacement in the Cbl riboswitchand identified analogous cryptic sites in structurally unrelated RNAsfrom HIV-1 and HCV. Finally, a ligand previously identified to targetthe env8 cryptic site bound both viral RNAs and yieldeddocking poses consistent with engagement of the newly exposed bindingsurfaces. Together, these results indicate that cryptic RNA bindingsites can be both energetically accessible and chemically addressable,expanding the range of conformational states that may contribute toRNA ligandability.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 18","pages":"2829–2834"},"PeriodicalIF":2.7,"publicationDate":"2026-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148964550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Unique Platform to Study the Dynamic Process of Prion Formation 研究朊病毒形成动态过程的独特平台

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-09-15 Epub Date: 2026-08-25 DOI: 10.1021/acs.biochem.6c00351

Jessica de Alcantara Ferreira, Daniel J. Walsh, Evelyn Turnbaugh, Jeremy H. Mills, Surachai Supattapone

{"title":"A Unique Platform to Study the Dynamic Process of Prion Formation","authors":"Jessica de Alcantara Ferreira, Daniel J. Walsh, Evelyn Turnbaugh, Jeremy H. Mills, Surachai Supattapone","doi":"10.1021/acs.biochem.6c00351","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00351","url":null,"abstract":"The pathogenic conversion of the cellular prion protein(PrPC) into the β-sheet-rich isoform PrPSc isthe pivotal pathogenic event in prion disease, yet the molecular stepsthat govern this structural transition remain elusive. In this study,we introduce a new approach to monitor site-specific conformationaltransitions that occur during infectious prion formation. The methodrelies on genetically encoded substitution of a fluorescent, environmentallysensitive noncanonical amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine(7-HCAA), into recombinant PrP substrate molecules, allowing real-timemonitoring of structural changes in high-efficiency in vitro PrPSc conversion reactions. As proof of principle, weshow that the W99 7-HCAA recPrP substrate efficiently propagates twodifferent PrPSc conformers (infectious cofactor PrPSc and noninfectious protein-only PrPSc). Bioassaysin knock-in mice expressing bank vole PrP confirm that W99 7-HCAAcofactor PrPSc produced by serial propagation is infectious,causing scrapie with an incubation period and neuropathological profilelike those induced by wild-type cofactor PrPSc. Markeddifferences in fluorescence intensity were observed between native,misfolded, and denatured states of W99 7-HCAA PrP, confirming that7-HCAA reports on local changes in PrP conformation. Together, thesefindings establish 7-HCAA as a site-specific and sensitive probe oflocal PrP conformation. Moreover, the results suggest a new strategyfor studying conformational dynamics in amyloid-forming proteins.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 18","pages":"2841–2850"},"PeriodicalIF":2.7,"publicationDate":"2026-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148964595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Programmable Enhancement of IRES-Mediated Translation via RNA-Guided eIF4G Recruitment 通过rna引导的eIF4G招募可编程增强ires介导的翻译

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-09-15 Epub Date: 2026-08-28 DOI: 10.1021/acs.biochem.6c00420

Binbin Ying, Feiyang Zheng, Yoshinori Kawabe, Masamichi Kamihira

{"title":"Programmable Enhancement of IRES-Mediated Translation via RNA-Guided eIF4G Recruitment","authors":"Binbin Ying, Feiyang Zheng, Yoshinori Kawabe, Masamichi Kamihira","doi":"10.1021/acs.biochem.6c00420","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00420","url":null,"abstract":"Internal ribosomeentry sites (IRESs) enable cap-independent translationbut often exhibit low and variable efficiency, limiting their usein synthetic gene circuits. Here, we present a programmable strategyto enhance IRES-dependent translation by recruiting a truncated eIF4Gscaffold via engineered RNA-binding protein fusions. A λN–eIF4Gfusion selectively increased EMCV IRES-mediated translation withoutaffecting cap-dependent expression, achieving up to 7.7-fold enhancement.Translation output was tunable over a broad dynamic range througheffector dosage and linker design, revealing key design parametersfor controlling activity. The system functioned across multiple mammaliancell lines and in tricistronic constructs, enabling coordinated regulationof polycistronic expression. Computational modeling was consistentwith preferential interaction between λN and the EMCV IRES.These results establish a modular framework for the programmable controlof translation, define design principles for RNA-guided translationalcontrol, and expand the synthetic biology toolkit for post-transcriptionalgene regulation.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 18","pages":"2905–2915"},"PeriodicalIF":2.7,"publicationDate":"2026-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148964613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Supramolecular Peptide Assemblies Efficiently Catalyze the Retroaldol Reaction 超分子肽组件有效催化后醛醇反应

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-09-15 Epub Date: 2026-08-31 DOI: 10.1021/acs.biochem.6c00380

Charitha J. Abeygunawardena, Liam R. Marshall, Ivan V. Korendovych

引用次数: 0

Membrane Models and the Pursuit of Coherence 膜模型和连贯性的追求。

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-09-15 Epub Date: 2026-07-22 DOI: 10.1021/acs.biochem.6c00367

Troy A. Kervin, Ziyao Zhang, Matthew Clark, Michael Overduin, Peijun Zhang

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Recent Advancements in Phospholipid Metabolic Labeling Using Clickable Probes 使用可点击探针进行磷脂代谢标记的最新进展

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-09-15 Epub Date: 2026-08-27 DOI: 10.1021/acs.biochem.6c00535

Robert J. Maraski, Michael D. Best

{"title":"Recent Advancements in Phospholipid Metabolic Labeling Using Clickable Probes","authors":"Robert J. Maraski, Michael D. Best","doi":"10.1021/acs.biochem.6c00535","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00535","url":null,"abstract":"Herein, we presenta perspective on recent advancementsin thefield of metabolic labeling as a method for interrogating phospholipidsin terms of their biosynthesis, trafficking, and localization. Phospholipidsare crucial for establishing cell morphology and signaling processesmediated by interactions between nearly all relevant cellular substrates.Bioorthogonal chemistry has provided a convenient platform in whichclickable probes that mimic biologically active precursors are deployedto generate labeled versions of phospholipids in cells, enabling thestudy of downstream lipid activities without affecting cell viability.The introduction of a clickable handle onto precursor analogs enablesvaried postderivatization techniques such as visualization with fluorescencemicroscopy, enhancement of mass spectrometric detection, as well asenrichment and identification following biotinylation, just to namea few. In this perspective, we outline how the sum of these studieshas provided a strong chemical toolbox for investigating lipid interactionsand functions within a cellular environment, enabling a deeper understandingof these indispensable biomolecules.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 18","pages":"2820–2828"},"PeriodicalIF":2.7,"publicationDate":"2026-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148964630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

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Thermodynamic Insights into the WT and Y220C TP53 DBDs Reveal that the Oncogenic Y220C Variant is a Loss-of-Function Mutation for Zn2+-Binding at Physiological Temperature 对WT和Y220C TP53 DBDs的热力学研究表明，致癌的Y220C变异是生理温度下Zn2+结合的功能缺失突变

IF 2.7 3区生物学

Biochemistry Biochemistry Pub Date : 2026-09-15 Epub Date: 2026-09-01 DOI: 10.1021/acs.biochem.6c00068

Jacob I. Stuckey, Valerie Vivat, Bradley M. Dickson, Jeremy W. Setser, Yong Li, Claudia M. Cremers, Jonathan E. Wilson, Daniele Peterle, John R. Engen, Thomas E. Wales, Rainer Wilcken, Erin B. Rohan, Gregg Chenail, James E. Audia, Robert J. Sims III

{"title":"Thermodynamic Insights into the WT and Y220C TP53 DBDs Reveal that the Oncogenic Y220C Variant is a Loss-of-Function Mutation for Zn2+-Binding at Physiological Temperature","authors":"Jacob I. Stuckey, Valerie Vivat, Bradley M. Dickson, Jeremy W. Setser, Yong Li, Claudia M. Cremers, Jonathan E. Wilson, Daniele Peterle, John R. Engen, Thomas E. Wales, Rainer Wilcken, Erin B. Rohan, Gregg Chenail, James E. Audia, Robert J. Sims III","doi":"10.1021/acs.biochem.6c00068","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00068","url":null,"abstract":"Wepresent evidence of previously unrecognized allostericconnectivityacross the TP53 DNA-binding domain (DBD). Specifically, we have foundevidence of an explicit influence on the Zn2+-binding sitefrom the region surrounding the hotspot Y220C mutation. This allostericconnectivity is intertwined with a temperature-dependent destabilizationof Zn2+ binding in both the WT and the Y220C DBDs. Ourstudies indicate that the Y220C mutation exacerbates this temperature-dependentdestabilization of Zn2+ binding to result in overall destabilizationof the Y220C variant. We provide detailed thermodynamic evidence thatRezatapopt, a small molecule reactivator of the Y220C DBD, engagesY220C through two thermodynamically distinct binding mechanisms andrestores WT-level Zn2+-affinity to this oncogenic variant.A series of thermodynamic models describing the WT and Y220C conformationallandscapes as well as the Rezatapopt binding mechanisms are proposed.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 18","pages":"2851–2864"},"PeriodicalIF":2.7,"publicationDate":"2026-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148964667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0