Biochemistry Biochemistry最新文献

{"title":"A Comparative Biophysical Study of Conserved Ubiquitin-like Protein Folds","authors":"Suhas Bhate,&nbsp;Neeraj Kumar Fauzdar,&nbsp;Kiran Sankar Chatterjee,&nbsp;Harika Kamma,&nbsp;Hemant Arya,&nbsp;Tarun Kumar Bhatt,&nbsp;Ranabir Das,&nbsp;Gajendra Singh","doi":"10.1021/acs.biochem.6c00263","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00263","url":null,"abstract":"<p>Ubiquitinand ubiquitin-like proteins (Ubls) constitute a conservedprotein family that shares a common β-grasp structural folddespite substantial sequence and functional divergence. How this highlyconserved fold is maintained while accommodating diverse biologicalfunctions remains an open question in protein structural biology.In this study, we provide atomistic insights into the structural featuresthat determine the stability of these conserved ubiquitin-like folds.Using an integrated biophysical approach combining solution NMR spectroscopy,molecular dynamics simulations, and chemical denaturation experiments,we systematically compare the protein dynamics and thermodynamic stabilityof ubiquitin and representative Ubls, including SUMO1, SUMO2, NEDD8,and Hub1. Our results reveal that ubiquitin exhibits exceptional thermodynamicstability relative to other Ubls, whereas Ubls display a broad rangeof stability profiles without a uniform trend toward destabilization.Biophysical and molecular dynamics analyses show that the reducedstability of Ubls is not associated with the loss of the conservedfold but is accompanied by differences in local structural flexibility.Together, these findings indicate that the conserved ubiquitin foldhas been maintained despite sequence divergence and variation in stabilityand dynamics, supporting functional diversity while preserving structuralintegrity.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3389–3398"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149306073","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}

{"title":"Thermophilic Adaptation of a Minimal Hammerhead Ribozyme Comes with a Functional Penalty","authors":"Ahmed H. Zegher,&nbsp;Samina F. Hanif,&nbsp;Evan M. Kielbasinski,&nbsp;Claire R. Patterson,&nbsp;Carrie Baker,&nbsp;Rigel P. Hall,&nbsp;Kathleen A. Leamy","doi":"10.1021/acs.biochem.6c00551","DOIUrl":"10.1021/acs.biochem.6c00551","url":null,"abstract":"<p>Life has adaptedto extreme environments, such as elevated pH,extreme temperatures, and environments with a lack of oxygen. Thebiomolecules made by organisms that live in extreme environments mustadapt to remain folded and functional. Recent computational studieshave shown that rRNA, tRNA, and certain mRNAs adapt in thermophilicorganisms through an increased GC content. However, few previous studieshave assessed the efficiency of adapted thermophilic RNAs. The workpresented herein uses minimal hammerhead ribozymes as a model systemto determine the effects of high-temperature adaptation, through increasedGC base pairing in the ribozyme secondary structure. Two hammerheadribozymes were designed based upon the minimal hammerhead 16 (HH16)ribozyme, a well-characterized model system. The mesophilic ribozymehas a moderate GC content (44%) in stems I, II, and III, while thethermophilic ribozyme has nearly double the GC content (76%) in thosesame stems. Both ribozymes share a conserved catalytic core and loop.Thermal denaturation experiments show that the model thermophilicribozyme is more thermostable with a higher single (cooperative) meltingtemperature than the model mesophilic ribozyme. However, thermostabilityis accompanied by a functional penalty. While at certain temperatures,the thermophilic ribozyme reaches the lower observed rates of themesophilic ribozyme, the thermophilic ribozyme never reaches the samemaximal rate of catalysis as the mesophilic ribozyme.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3409–3418"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149222940","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}

{"title":"EF-Hand Binding Order and Conformational Control of Lanthanide Ion Binding in M. extorquens Lanmodulin","authors":"Claire C. Mammoser,&nbsp;Natalie A. Savastio,&nbsp;Joseph A. Cotruvo, Jr.","doi":"10.1021/acs.biochem.6c00566","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00566","url":null,"abstract":"<p>Biomolecular chelatorshave emerged recently as potentialalternativesto conventional, energy-intensive methods for the challenging separationof rare earth elements (REEs), including the lanthanide (Ln^III) ions. Lanmodulin (LanM) from <em>Methylobacterium extorquens</em> is the archetype for highly selective biological REE chelation andhas been applied for recovery and separations of REEs. LanM’shigh affinity and selectivity are driven by a large disorder-to-orderconformational transition in response to cooperative binding of Ln^III ions to three of its EF-hand motifs, with one site beingmore labile than the other two. Definition of the order of Ln^III ion binding and the structural interactions contributingto cooperativity has been elusive but is essential for engineeringof LanM and other REE-binding proteins. Here, we use a combinationof mutagenesis and spectroscopic methods (UV absorbance, circulardichroism, luminescence, and NMR spectroscopies) to achieve both aims.We identify a salt bridge (K93-D56) between LanM’s two primaryhelices, disruption of which inactivates one of the metal-bindingsites. Together, our results suggest, contrary to previous hypotheses,that EF1 and EF2 are the tightest sites, which are sufficient to organizemost of the protein’s tertiary structure; EF3 is the labile,third binding site; and REEs, circumneutral pH, and the salt bridgeare all essential for LanM’s full conformational change. Theseresults help to define the conformational landscape underlying LanM’sselective REE recognition and provide design principles for engineeringREE-binding proteins for recovery and separation applications.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3501–3513"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149305633","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}

{"title":"In Vitro Reconstitution of Vertebrate Sonic Hedgehog Protein Cholesterolysis; Native and Engineered Sterol Specificity","authors":"Dayton C. Seidel,&nbsp;Andrew G. Wagner,&nbsp;John L. Pezzullo,&nbsp;Katherine A. Thayer,&nbsp;Seth Beadle,&nbsp;Margot L. Olejarczyk,&nbsp;Logan R. Bartlett,&nbsp;José-Luis Giner,&nbsp;Brian P. Callahan","doi":"10.1021/acs.biochem.6c00321","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00321","url":null,"abstract":"<p>Extracellular secretion of the oncogenic sonic hedgehogsignalingligand is contingent on release from a precursor protein through peptide-bondcholesterolysis, mediated by the hedgehog C-terminal domain, SHhC.In this work, we first describe the in vitro reconstitution of SHhCdomains from vertebrate model organisms, <em>Xenopus laevis</em> (Xla) and <em>Danio rerio</em> (Dre). Cholesterolysisis assayed continuously in multiwell plates by monitoring changesin fluorescence resonance energy transfer (FRET) from a bacteriallyexpressed, engineered precursor construct. Using this FRET assay,we found that Xla and Dre SHhC exhibit high substrate stereospecificity,accepting cholesterol (<em>K</em>_M, 1–2μM, cholesterolysis <em>t</em>_1/2 of ∼11min) while rejecting the 3-alpha epimer, epi-cholesterol (<em>K</em>_M &gt; 100 μM, <em>t</em>_1/2 &gt; 10 h). Next, screening a 96-member detergent/surfactantlibraryfor compatibility with SHhC activity, we observed a shared preferencefor the zwitterionic n-dodecylphosphocholine (DPC, Fos-choline-12),while finding that cationic detergents appeared incompatible withcholesterolysis. Lastly, we report that an alanine point mutationat a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC enablesengineering of substrate specificity away from cholesterol and towardrationally designed hypernucleophilic sterols via chemical rescue.Of those sterols, 2-β-carboxycholestanol was active as a substratewith D46A variants only; the remaining sterols were accepted by bothD46A and wild-type SHhC. In summary, we have established a FRET-basedkinetic assay to continuously monitor enzymatic activity of vertebrateSHhC domains, a key step toward pharmacological manipulation of Sonichedgehog protein biosynthesis, while also identifying the first SHhCmutant/sterol pairing for orthogonal sterolysis.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3533–3547"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149305646","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}

{"title":"Covalent Labeling of Human Neuroglobin with a 19F-Probe to Study its Conformational Changes Using 19F NMR","authors":"Si-Qi Xia,&nbsp;Xi-Chun Liu,&nbsp;Shu-Qin Gao,&nbsp;Hong Yuan,&nbsp;Xiangshi Tan,&nbsp;Ying-Wu Lin","doi":"10.1021/acs.biochem.6c00592","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00592","url":null,"abstract":"<p>It is of interest to develop new approaches to probingproteinconformational changes and protein–protein interactions. Inthis study, we covalently labeled human neuroglobin (Ngb) with a ¹⁹F-probe containing a Ph-OCF₃ group at Cys120 andby introducing cysteine residues at specific positions in the C120SNgb mutant. ¹⁹F NMR provides valuable insights into itsconformational changes upon cleavage of the intramolecular disulfidebond of Cys46-Cys55, as well as its interactions with protein partnerssuch as cytochrome <em>c</em> (Cyt <em>c</em>) throughthe formation of the Ngb-Cyt <em>c</em> complex. This studypresents a convenient approach to investigating the dynamic propertiesof heme globins regulated by disulfide bonds and protein–proteininteractions.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3371–3374"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149306071","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}

{"title":"Thermodynamic and Structural Consequences of Nucleotide Deletion in DNA/RNA Heteroduplexes","authors":"Tomoka Akita,&nbsp;Taiichi Sakamoto,&nbsp;Elisa Tomita-Sudo,&nbsp;Renshin Sano,&nbsp;Nae Sakimoto,&nbsp;Shigenori Iwai,&nbsp;Satoshi Obika,&nbsp;Takao Inoue,&nbsp;Junji Kawakami","doi":"10.1021/acs.biochem.6c00502","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00502","url":null,"abstract":"<p>Nucleotide deletions in nucleic acid duplexes can inducenoncanonicalstructures such as bulges and terminal fraying, which may influencethe off-target effects of antisense oligonucleotides (ASOs). However,the thermodynamic and structural properties of deletion-containingDNA/RNA heteroduplexes remain poorly understood. In this study, wesystematically investigated the effects of single-nucleotide deletionsat different positions in a twenty-mer DNA strand hybridized withcomplementary RNA. Thermodynamic parameters were determined from UVmelting analyses, and structural changes were examined using NMR iminoproton spectroscopy. All deletion-containing duplexes exhibited reducedthermodynamic stability relative to that of the fully matched duplex.The extent of destabilization strongly depended on the deletion position:terminal deletions caused only modest destabilization, whereas deletionsnear the central region produced substantially larger energetic penalties.The largest difference in stability among the duplexes correspondedto an approximately 10³-fold difference in binding constants.These results suggest a structural transition between terminal frayingand internal bulge formation. Central deletions likely induce structuralstrain and disruption of base-stacking interactions, whereas terminaldeletions can be accommodated through fraying at the helix ends. NMRanalyses further revealed that structural perturbations caused bynucleotide deletion were not always localized to the deletion site.In several duplexes, disappearance of imino proton signals was observedat positions a few nucleotides away from the deletion, indicatinglong-range propagation of structural instability and reduced helicalcooperativity. These findings provide new insights into the structuralstability of DNA/RNA heteroduplexes and provide a fundamental physicochemicalframework for understanding deletion-containing DNA/RNA heteroduplexesand for evaluating their potential relevance to off-target interactionsin ASO systems.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3477–3485"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149305702","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}

{"title":"Indirect Readout Drives Cre-loxP Recognition Thermodynamics","authors":"Jonathan S. Montgomery,&nbsp;Megan E. Judson,&nbsp;Robert M. Bauer,&nbsp;Matthew J. Benedek,&nbsp;Caroline E. Hervey,&nbsp;Mark P. Foster","doi":"10.1021/acs.biochem.6c00409","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00409","url":null,"abstract":"<p>Cre, a site-specific tyrosine DNA recombinase, enablestargetedmanipulation of genetic material without the production of cytotoxicdouble-strand DNA breaks. Though widely deployed in biotechnology,an incomplete understanding of how Cre recognizes its cognate <em>loxP</em> target limits its broader application in human health.Cre is highly specific for its cognate loxP sites, comprising an invertedpair of recombinase binding elements (RBEs) and an asymmetric spacer.Structural studies have shown that Cre protomers make few direct base-specificcontacts to each RBE, suggesting that specificity arises through indirectreadout, wherein noncognate sites are unable to adopt favorable binding-coupledconformations. We designed a set of <em>loxP</em> half-sitevariants predicted to alter local DNA shape characteristics, includingminor-groove geometry and base-step parameters, and measured the thermodynamicsof Cre binding by isothermal titration calorimetry. Variable-temperaturethermograms and nuclear magnetic resonance spectra reveal that unfavorablemutations at noncontacted positions in the RBE reduce signatures ofbinding-coupled conformational changes. Moreover, mass photometryof Cre binding to oligonucleotides containing two properly spacedRBEs revealed that noncontacted positions in the intervening 8-basepair spacer influence cooperative dimerization of two Cre protomersat <em>loxP</em> sites and their synapsis to form catalyticallyactive Cre₄-<em>loxP</em>₂ complexes.These results demonstrate that noncontacted positions contribute tospecificity by encoding favorable DNA mechanics, offering new designprinciples for engineering Cre variants that target alternative DNAsequences.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3399–3408"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149305873","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}

{"title":"TP-Ring and ZN-Ring Systems Allow Efficient Protein Clamping, Solubilization and Stabilization","authors":"Jade Wenzel,&nbsp;Alexandre Kriznik,&nbsp;Marie-Eve Chagot,&nbsp;Marc Quinternet","doi":"10.1021/acs.biochem.6c00444","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00444","url":null,"abstract":"<p>Improvingrecombinant protein stability remains a centralchallengein biotechnology, as many proteins exhibit limited robustness duringproduction and use. Here, we introduce two intramolecular “clamping”systems, termed ZN-ring and TP-ring, that exploit ultrastable noncovalentprotein–protein interactions to constrain the N- and C-terminiof a target protein. These systems are derived from the high-affinitycomplexes formed by ZNHIT3/NUFIP1 and TAH1/PIH1, respectively, whoseinteractions are driven by extensive hydrophobic interfaces. Proof-of-conceptconstructs demonstrate that intrachain clamping preserves native-likeinteractions and yields highly stable assemblies with melting temperaturesexceeding 95 °C. When applied to a model protein, the TP-ringsignificantly enhances thermal stability, consistent with structuralpredictions suggesting additional stabilizing contacts. Furthermore,clamping markedly improves the soluble expression of a challengingpolyethylene terephthalate hydrolase (PETase) in <em>Escherichia coli</em>, without compromising its catalytically activefold. These results establish protein clamping as a versatile strategyto enhance stability, solubility, and expression. Given their intrinsicrobustness and broad compatibility with diverse targets, ZN- and TP-ringsystems provide a promising platform for protein engineering, withpotential applications in biotechnology and structural biology.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3375–3382"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149305945","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}

{"title":"Randomization of the Damaged Base-Capping Loop Rewires Substrate Specificity in Formamidopyrimidine–DNA Glycosylase","authors":"Nikita A. Bulgakov,&nbsp;Margarita M. Amanova,&nbsp;Anton V. Endutkin,&nbsp;Kristina A. Makasheva,&nbsp;Franck Coste,&nbsp;Bertrand Castaing,&nbsp;Victor M. Golyshev,&nbsp;Alexander A. Lomzov,&nbsp;Anna V. Yudkina,&nbsp;Dmitry O. Zharkov","doi":"10.1021/acs.biochem.6c00579","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00579","url":null,"abstract":"<p>Formamidopyrimidine–DNAglycosylase (Fpg) is a bacterialbase excision repair enzyme that removes 8-oxoguanine (8-oxoG) andsome other oxidatively damaged nucleobases from DNA. Fpg hydrolyzesthe <em>N</em>-glycosidic bond of the damaged nucleotide andnicks DNA at the resulting apurinic/apyrimidinic (AP) site by β-elimination.Together with bacterial endonuclease VIII and eukaryotic homologues,more specific for damaged pyrimidines, Fpg belongs to a helix–two-turn–helix(H2TH) superfamily of DNA glycosylases. Crystal structures of Fpgreveal that the enzyme recognizes 8-oxoG by flipping it out of theDNA duplex and capping it with a flexible tetrapeptide loop. However,this loop is poorly conserved and contacts 8-oxoG only through themain-chain amides, raising questions about the enzyme specificitydeterminants. To clarify the mechanism of 8-oxoG recognition, we haveproduced and characterized five variants of Fpg from <em>Lactococcus lactis</em> with a randomized capping loop. All of these mutantswere deficient in 8-oxoG repair, both <em>in vitro</em> andin the <em>E. coli</em> complementation assay, yet retainedthe ability to bind AP site-containing DNA, form a Schiff base covalentreaction intermediate, and catalyze β-elimination. The mutantswere still active in the removal of 5-hydroxyuracil (OHU), an alternativeFpg substrate. All loop mutants showed relaxed opposite-base specificity,processing AP sites and OHU opposite either C or A. Thus, whereasthe general architecture of the active site in H2TH DNA glycosylasesis permissive for the excision of a wide range of oxidized bases,the capping loop endows Fpg with the specificity for its biologicallyrelevant substrate, 8-oxoguanine.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3419–3433"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149305722","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}

{"title":"Histidine-51 Facilitates the Conformational Change and Deprotonation of Horse Liver Alcohol Dehydrogenase–NAD+ Complexes","authors":"Bryce V. Plapp","doi":"10.1021/acs.biochem.6c00532","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00532","url":null,"abstract":"<p>Structural studiessuggest that His-51 in wild-type horse liveralcohol dehydrogenase (ADH1E) facilitates proton transfer betweenthe hydroxyl group of a ligand bound to the catalytic zinc and bulksolvent. Previous work showed that the Gln-51 substitution increasesthe p<em>K</em> values for pH dependencies for various enzymaticreactions by about 1.5 units, and stopped-flow studies on the bindingof NAD⁺ and pyrazole suggested that the equilibrium positionfor the global conformational change of the enzyme–NAD⁺ complex became disfavored by 1000-fold. That conclusion neededconfirmation. Here, X–ray crystallography of six ternary complexesof H51Q ADH with NAD⁺ and pyrazole, caprate, 2,2,2-trifluoroethanol,or 2,3,4,5,6-pentafluorobenzyl alcohol, and NADH and water or <em>N</em>-cyclohexylformamide shows that all complexes have thesame closed conformation as wild-type ADH. Gln-51 fits in the regionoccupied by His-51 and does not affect the positions of bound coenzymesor inhibitors that coordinate to the catalytic zinc. Stopped-flowstudies show that the rate-limiting formation of ternary complexeswith H51Q ADH and NAD⁺ depends upon deprotonation of theligands that bind to the catalytic zinc (pyrazole or 2,2,2-trifluoroethanol).Furthermore, the binding of NAD⁺ and trifluoroethanol monitoredby fluorescence and proton release show that the H51Q substitutiondisfavors the conformational change by about 3-fold as compared towild-type ADH. The p<em>K</em> values of the enzyme–NAD⁺ complexes with water and other zinc ligands increase becauseHis-51 no longer facilitates the deprotonation. The roles of the zinc-waterand His-51 in the mechanism are exposed.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 21","pages":"3486–3500"},"PeriodicalIF":2.7,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149305860","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}