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The β Subunit Loop That Couples Catalysis and Rotation in ATP Synthase Has a Critical Length*

Mnatsakanyan, Nelli; Kemboi, Silas K.; Salas, Jasmin; Weber, Joachim
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix structure in the C-terminal domain of the β subunit containing the conserved DELSEED motif, termed “DELSEED-loop,” was suggested to be involved in coupling between catalysis and rotation. If this is indeed the role of the loop, it must have a critical length, the minimum length required to sustain its function. Here, the critical length of the DELSEED-loop was determined by functional analysis of mutants of Bacillus PS3 ATP synthase that had 7–14 amino acids within the loop deleted. A 10 residue deletion lost the ability to catalyze ATP synthesis, but was still an active ATPase. Deletion of 14 residues abolished any enzymatic activity. Modeling indicated that in both deletion mutants the DELSEED-loop was shortened by ∼10 Å; fluorescence resonance energy transfer experiments confirmed the modeling results. This appears to define the minimum length for DELSEED-loop required for coupling of catalysis and rotation. In addition, we could demonstrate that the loss of high-affinity binding to the catalytic site(s) that had been observed previously in two deletion mutants with 3–4 residues removed was not due to the loss of negative charged residues of the DELSEED motif in these mutants. An AALSAAA mutant in which all negative charges of the DELSEED motif were removed showed a normal pattern for MgATP binding to the catalytic sites...

An Active Site Guanine Participates in glmS Ribozyme Catalysis in its Protonated State

Viladoms, Júlia; Scott, Lincoln G.; Fedor, Martha J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Active site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2′-OH nucleophile. Apparent pKa values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active site guanine, in general base catalysis. Furthermore, discrepancies between apparent pKa values obtained from functional assays and microscopic pKa values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analog, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form, donates a hydrogen bond to stabilize the transition state.

Flexibility, Diversity, and Cooperativity: Pillars of Enzyme Catalysis

Hammes, Gordon G.; Benkovic, Stephen J.; Hammes-Schiffer, Sharon
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, NMR, and single molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme-substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conformations, and cooperative conformational changes are important features of this model.

Domain Analysis Reveals That a Deubiquitinating Enzyme USP13 Performs Non-Activating Catalysis for Lys63-Linked Polyubiquitin

Zhang, Yu-Hang; Zhou, Chen-Jie; Zhou, Zi-Ren; Song, Ai-Xin; Hu, Hong-Yu
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 28/12/2011 Português
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Deubiquitination is a reverse process of cellular ubiquitination important for many biological events. Ubiquitin (Ub)-specific protease 13 (USP13) is an ortholog of USP5 implicated in catalyzing hydrolysis of various Ub chains, but its enzymatic properties and catalytic regulation remain to be explored. Here we report studies of the roles of the Ub-binding domains of USP13 in regulatory catalysis by biochemical and NMR structural approaches. Our data demonstrate that USP13, distinct from USP5, exhibits a weak deubiquitinating activity preferring to Lys63-linked polyubiquitin (K63-polyUb) in a non-activation manner. The zinc finger (ZnF) domain of USP13 shares a similar fold with that of USP5, but it cannot bind with Ub, so that USP13 has lost its ability to be activated by free Ub. Substitution of the ZnF domain with that of USP5 confers USP13 the property of catalytic activation. The tandem Ub-associated (UBA) domains of USP13 can bind with different types of diUb but preferentially with K63-linked, providing a possible explanation for the weak activity preferring to K63-polyUb. USP13 can also regulate the protein level of CD3δ in cells, probably depending on its weak deubiquitinating activity and the Ub-binding properties of the UBA domains. Thus...

Chemical Synthesis of Complex Molecules Using Nanoparticle Catalysis

Cong, Huan; Porco, John A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/01/2012 Português
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Nanoparticle catalysis has emerged as an active topic in organic synthesis. Of particular interest is the development of enabling methodologies to efficiently assemble complex molecules using nanoparticle catalysis. This Viewpoint highlights recent developments and discusses future perspectives in this emerging field.

Arg375 Tunes Tetrahydrobiopterin Functions and Modulates Catalysis by Inducible Nitric Oxide Synthase

Wang, Zhi-Qiang; Tejero, Jesús; Wei, Chin-Chuan; Haque, Mohammad Mahfuzul; Santolini, Jerome; Fadlalla, Mohammed; Biswas, Ashis; Stuehr, Dennis J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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NO synthase enzymes (NOS) support unique single-electron transitions of a bound H4B cofactor during catalysis. Previous studies showed that both the pterin structure and surrounding protein residues impact H4B redox function during catalysis. A conserved Arg residue (Arg375 in iNOS) forms hydrogen bonds with the H4B ring. In order to understand the role of this residue in modulating the function of H4B and overall NO synthesis of the enzyme, we generated and characterized three mutants R375D, R375K and R375N of the oxygenase domain of inducible NOS (iNOSoxy). The mutations affected the dimer stability of iNOSoxy and its binding affinity towards substrates and H4B to varying degrees. Optical spectra of the ferric, ferrous, ferrous dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to the wild-type. However, mutants displayed somewhat lower heme midpoint potentials and faster ferrous heme-NO complex reactivity with O2. Unlike the wild-type protein, mutants could not oxidize NOHA to nitrite in a H2O2-driven reaction. Mutation could potentially change the ferrous dioxy decay rate, H4B radical formation rate, and the amount of the Arg hydroxylation during single turnover Arg hydroxylation reaction. All mutants were able to form heterodimers with the iNOS G450A full-length protein and displayed lower NO synthesis activities and uncoupled NADPH consumption. We conclude that the conserved residue Arg375 (1) regulates the tempo and extent of the electron transfer between H4B and ferrous dioxy species and (2) controls the reactivity of the heme-based oxidant formed after electron transfer from H4B during steady state NO synthesis and in H2O2-driven NOHA oxidation. Thus...

N-Heterocyclic carbene/Brønsted acid cooperative catalysis as a powerful tool in organic synthesis

De Vreese, Rob; D’hooghe, Matthias
Fonte: Beilstein-Institut Publicador: Beilstein-Institut
Tipo: Artigo de Revista Científica
Publicado em 14/03/2012 Português
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The interplay between metals and N-heterocyclic carbenes (NHCs) has provided a window of opportunities for the development of novel catalytic strategies within the past few years. The recent successful combination of Brønsted acids with NHCs has added a new dimension to the field of cooperative catalysis, enabling the stereoselective synthesis of functionalized pyrrolidin-2-ones as valuable scaffolds in heterocyclic chemistry. This Commentary will briefly highlight the concept of N-heterocyclic carbene/Brønsted acid cooperative catalysis as a new and powerful methodology in organic chemistry.

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

de Giuseppe, Priscila O.; Martins, Nadia H.; Meza, Andreia N.; dos Santos, Camila R.; Pereira, Humberto D’Muniz; Murakami, Mario T.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 05/09/2012 Português
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The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2′deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5′ hydroxyl group of adenosine and Arg43* side chain contributes for the ribosyl radical to adopt an unusual C3’-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl6 and Br8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore...

Loop Residues and Catalysis in OMP Synthase†

Wang, Gary P.; Hansen, Michael Riis; Grubmeyer, Charles
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Residue-to-alanine mutations and a two amino acid deletant have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, E.C. 2.4.2.10). As described previously, the K103A mutant enzyme showed a 104-fold decrease in kcat/KM for PRPP; K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 each gave a 40- and 7-fold decrease in kcat/KM, respectively, and E101A, D104A, G106A were slightly faster than WT in kcat with minor effects on kcat/KM. Equilibrium binding of OMP or PRPP in binary complexes was little affected by loop mutation, suggesting that the energetics of ground state binding have little contribution from the catalytic loop, or that favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 104-fold reduction of kcat but almost unaltered KD values for all four substrate molecules. The 20% (i.e. 1.20) intrinsic [1′-3H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast...

Role of Motif III in Catalysis by Acetyl-CoA Synthetase

Ingram-Smith, Cheryl; Thurman, Jerry L.; Zimowski, Karen; Smith, Kerry S.
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
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The acyl-adenylate-forming enzyme superfamily, consisting of acyl- and aryl-CoA synthetases, the adenylation domain of the nonribosomal peptide synthetases, and luciferase, has three signature motifs (I–III) and ten conserved core motifs (A1–A10), some of which overlap the signature motifs. The consensus sequence for signature motif III (core motif A7) in acetyl-CoA synthetase is Y-X-S/T/A-G-D, with an invariant fifth position, highly conserved first and fourth positions, and variable second and third positions. Kinetic studies of enzyme variants revealed that an alteration at any position resulted in a strong decrease in the catalytic rate, although the most deleterious effects were observed when the first or fifth positions were changed. Structural modeling suggests that the highly conserved Tyr in the first position plays a key role in active site architecture through interaction with a highly conserved active-site Gln, and the invariant Asp in the fifth position plays a critical role in ATP binding and catalysis through interaction with the 2′- and 3′-OH groups of the ribose moiety. Interactions between these Asp and ATP are observed in all structures available for members of the superfamily, consistent with a critical role in substrate binding and catalysis for this invariant residue.

Role of Protein Conformational Dynamics in the Catalysis by 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase†

Yan, Honggao; Ji, Xinhua
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/2011 Português
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Enzymatic catalysis has conflicting structural requirements of the enzyme. In order for the enzyme to form a Michaelis complex, the enzyme must be in an open conformation so that the substrate can get into its active center. On the other hand, in order to maximize the stabilization of the transition state of the enzymatic reaction, the enzyme must be in a closed conformation to maximize its interactions with the transition state. The conflicting structural requirements can be resolved by a flexible active center that can sample both open and closed conformational states. For a bisubstrate enzyme, the Michaelis complex consists of two substrates in addition to the enzyme. The enzyme must remain flexible upon the binding of the first substrate so that the second substrate can get into the active center. The active center is fully assembled and stabilized only when both substrates bind to the enzyme. However, the side-chain positions of the catalytic residues in the Michaelis complex are still not optimally aligned for the stabilization of the transition state, which lasts only approximately 10−13 s. The instantaneous and optimal alignment of catalytic groups for the transition state stabilization requires a dynamic enzyme, not an enzyme which undergoes a large scale of movements but an enzyme which permits at least a small scale of adjustment of catalytic group positions. This review will summarize the structure...

Dynamics connect substrate recognition to catalysis in protein kinase A

Masterson, Larry R.; Cheng, Cecilia; Yu, Tao; Tonelli, Marco; Kornev, Alexandr; Taylor, Susan S.; Veglia, Gianluigi
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Atomic resolution studies of protein kinases have traditionally been carried out in the inhibitory state, limiting our current knowledge on the mechanisms of substrate recognition and catalysis. Using NMR, x-ray crystallography, and thermodynamic measurements we analyzed the substrate recognition process of cAMP-dependent protein kinase (PKA), finding that entropy and protein dynamics play a prominent role. The nucleotide acts as a dynamic and allosteric activator by coupling the two lobes of apo PKA, enhancing the enzyme dynamics synchronously, and priming it for catalysis. The formation of the ternary complex is entropically driven and NMR spin relaxation data reveal that both substrate and PKA are dynamic in the closed state. Our results show that the enzyme toggles between open and closed states, which indicate that a population shift/conformational selection rather than an induced-fit mechanism governs substrate recognition.

Precursor of ether phospholipids is synthesized by a flavoenzyme through covalent catalysis

Nenci, Simone; Piano, Valentina; Rosati, Sara; Aliverti, Alessandro; Pandini, Vittorio; Fraaije, Marco W.; Heck, Albert J. R.; Edmondson, Dale E.; Mattevi, Andrea
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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The precursor of the essential ether phospholipids is synthesized by a peroxisomal enzyme that uses a flavin cofactor to catalyze a reaction that does not alter the redox state of the substrates. The enzyme crystal structure reveals a V-shaped active site with a narrow constriction in front of the prosthetic group. Mutations causing inborn ether phospholipid deficiency, a very severe genetic disease, target residues that are part of the catalytic center. Biochemical analysis using substrate and flavin analogs, absorbance spectroscopy, mutagenesis, and mass spectrometry provide compelling evidence supporting an unusual mechanism of covalent catalysis. The flavin functions as a chemical trap that promotes exchange of an acyl with an alkyl group, generating the characteristic ether bond. Structural comparisons show that the covalent versus noncovalent mechanistic distinction in flavoenzyme catalysis and evolution relies on subtle factors rather than on gross modifications of the cofactor environment.

Water Networks in Fast Proton Transfer During Catalysis by Human Carbonic Anhydrase II†

Mikulski, Rose; West, Dayne; Sippel, Katherine H.; Avvaru, Balendu Sankara; Aggarwal, Mayank; Tu, Chingkuang; McKenna, Robert; Silverman, David N.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Variants of human carbonic anhydrase II (HCA II) with amino-acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and 18O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing the replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for proton transfer from His64 to the zinc-bound hydroxide in catalysis were 4 μs-1 and 9 μs-1 for Y7F and Y7F-N67Q, respectively, compared with a value of 0.8 μs-1 for wild-type HCA II. These higher values observed for Y7F and Y7F-N67Q HCA II could not be explained by differences in the values of the pKa of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with reduced branching in the networks of hydrogen-bonded water molecules between the proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5 – 1.6 Å resolution. Moreover, Y7F-N67Q HCA II is unique among the variants studied in having a direct...

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme

Biondi, Elisa; Poudyal, Raghav R.; Forgy, Joshua C.; Sawyer, Andrew W.; Maxwell, Adam W. R.; Burke, Donald H.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu2+ for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu2+, indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu2+ binding by RNA; rather, these data establish that Cu2+ enters the active site within a Cu2+•GTPγS or Cu2+•GTP chelation complex, and that Cu2+•nucleobase interactions further enforce Cu2+ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg2+ with [Co(NH3)6]3+ significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg2+ coordination is structural rather than catalytic. Replacing Mg2+ with alkaline earths of increasing ionic radii (Ca2+, Sr2+ and Ba2+) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1...

Catalysis in the Service of Green Chemistry: Nobel Prize-Winning Palladium-Catalysed Cross-Couplings, Run in Water at Room Temperature: Heck, Suzuki-Miyaura and Negishi reactions carried out in the absence of organic solvents, enabled by micellar catalysis

Lipshutz, Bruce H.; Taft, Benjamin R.; Abela, Alexander R.; Ghorai, Subir; Krasovskiy, Arkady; Duplais, Christophe
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/2012 Português
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Palladium-catalysed cross-couplings, in particular Heck, Suzuki-Miyaura and Negishi reactions developed over three decades ago, are routinely carried out in organic solvents. However, alternative media are currently of considerable interest given an increasing emphasis on making organic processes ‘greener’; for example, by minimising organic waste in the form of organic solvents. Water is the obvious leading candidate in this regard. Hence, this review focuses on the application of micellar catalysis, in which a ‘designer’ surfactant enables these award-winning coupling reactions to be run in water at room temperature.

The RNA polymerase bridge helix YFI motif in catalysis, fidelity and translocation

Nedialkov, Yuri A.; Opron, Kristopher; Assaf, Fadi; Artsimovitch, Irina; Kireeva, Maria L.; Kashlev, Mikhail; Cukier, Robert I.; Nudler, Evgeny; Burton, Zachary F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The bridge α-helix in the β′ subunit of RNA polymerase (RNAP) borders the active site and may have roles in catalysis and translocation. In Escherichia coli RNAP, a bulky hydrophobic segment near the N-terminal end of the bridge helix is identified (β′ 772-YFI-774; the YFI motif). YFI is located at a distance from the active center and adjacent to a glycine hinge (β′ 778-GARKG-782) involved in dynamic bending of the bridge helix. Remarkably, amino acid substitutions in YFI significantly alter intrinsic termination, pausing, fidelity and translocation of RNAP. F773V RNAP largely ignores the λ tR2 terminator at 200 µM NTPs and is strongly reduced in λ tR2 recognition at 1 µM NTPs. F773V alters RNAP pausing and backtracking and favors misincorporation. By contrast, the adjacent Y772A substitution increases fidelity and exhibits other transcriptional defects generally opposite to those of F773V. All atom molecular dynamics simulation revealed two separate functional connections emanating from YFI explaining the distinct effects of substitutions: Y772 communicates with the active site through the link domain in the β subunit, whereas F773 communicates through the fork domain in the β subunit. I774 interacts with the F-loop...

Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification

Yakovleva, Lyudmila; Shuman, Stewart
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3′-phosphotyrosyl274)-enzyme intermediate. Conserved active site side chains promote the attack of Tyr274 on the scissile phosphodiester via transition state stabilization and general acid catalysis. Two essential side chains, Lys167 and Arg130, act in concert to protonate and expel the 5′-O leaving group. Here we gained new insights to catalysis through chemical mutagenesis of Lys167. Changing Lys167 to cysteine crippled the DNA cleavage and religation transesterification steps (kcl = 4.3 × 10−4 s−1; krel = 9 × 10−4 s−1). The transesterification activities of the K167C enzyme were revived by in vitro alkylation with 2-bromoethylamine (kcl = 0.031 s−1; krel ≥ 0.4 s−1) and 3-bromopropylamine (kcl = 0.013 s−1; krel = 0.22 s−1), which convert the cysteine to γ-thialysine and γ-thiahomolysine, respectively. These chemically installed lysine analogs were more effective than a genetically programmed arginine-167 substitution characterized previously. The modest differences in the transesterification rates of the 2-bromoethylamine and 3-bromopropylamine-treated enzymes highlights that TopIB is tolerant of a longer homolysine side chain for assembly of the active site and formation of the transition state.

Vitamin K Oxygenation, Glutamate Carboxylation, and Processivity: Defining the Three Critical Facets of Catalysis by the Vitamin K–Dependent Carboxylase12

Rishavy, Mark A.; Berkner, Kathleen L.
Fonte: American Society for Nutrition Publicador: American Society for Nutrition
Tipo: Artigo de Revista Científica
Publicado em 02/03/2012 Português
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The vitamin K–dependent carboxylase uses vitamin K oxygenation to drive carboxylation of multiple glutamates in vitamin K–dependent proteins, rendering them active in a variety of physiologies. Multiple carboxylations of proteins are required for their activity, and the carboxylase is processive, so that premature dissociation of proteins from the carboxylase does not occur. The carboxylase is unique, with no known homology to other enzyme families, and structural determinations have not been made, rendering an understanding of catalysis elusive. Although a model explaining the relationship of oxygenation to carboxylation had been developed, until recently almost nothing was known of the function of the carboxylase itself in catalysis. In the past decade, discovery and analysis of naturally occurring carboxylase mutants has led to identification of functionally relevant residues and domains. Further, identification of nonmammalian carboxylase orthologs has provided a basis for bioinformatic analysis to identify candidates for critical functional residues. Biochemical analysis of rationally chosen carboxylase mutants has led to breakthroughs in understanding vitamin K oxygenation, glutamate carboxylation, and maintenance of processivity by the carboxylase. Protein carboxylation has also been assessed in vivo...

DNA Catalysis of a Normally Disfavored RNA Hydrolysis Reaction

Parker, Darren J.; Xiao, Ying; Aguilar, John M.; Silverman, Scott K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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We recently used in vitro selection to identify many deoxyribozymes that catalyze DNA phosphodiester bond hydrolysis and create 5′-phosphate and 3′-hydroxyl termini. Alternatively, numerous deoxyribozymes have been identified for catalysis of RNA cleavage by 2′-hydroxyl transesterification, forming 2′,3′-cyclic phosphate and 5′-hydroxyl termini. In this study, we investigated the ability of DNA to catalyze RNA cleavage by hydrolysis rather than transesterification, although normally the hydrolysis reaction is substantially disfavored relative to transesterification. Via a series of in vitro selection experiments, we found that reselection of a DNA-hydrolyzing deoxyribozyme leads either to transesterification or hydrolysis, depending on exclusion or inclusion of a stringent selection pressure for hydrolysis. An entirely new selection starting from a random DNA pool, using an all-RNA substrate and imposing the same selection pressure, also leads to RNA hydrolysis. Collectively, these results establish experimentally that small DNA sequences have the catalytic ability to direct a chemical reaction down a disfavored pathway, even when a more favorable mechanism is readily available. Our view of DNA catalysis is therefore expanded beyond merely increasing the rates of reactions that would have occurred more slowly without the catalyst.