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Amino Acid Residues in the GIY-YIG Endonuclease II of Phage T4 Affecting Sequence Recognition and Binding as Well as Catalysis▿ †

Lagerbäck, Pernilla; Carlson, Karin
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76...

Mechanistic characterization of the HDV genomic ribozyme: The cleavage site base pair plays a structural role in facilitating catalysis

Cerrone-Szakal, Andrea L.; Chadalavada, Durga M.; Golden, Barbara L.; Bevilacqua, Philip C.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /09/2008 Português
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The hepatitis delta virus (HDV) ribozyme occurs in the genomic and antigenomic strands of the HDV RNA and within mammalian transcriptomes. Previous kinetic studies suggested that a wobble pair (G•U or A+•C) is preferred at the cleavage site; however, the reasons for this are unclear. We conducted sequence comparisons, which indicated that while G•U is the most prevalent combination at the cleavage site, G-C occurs to a significant extent in genomic HDV isolates, and G•U, G-C, and A-U pairs are present in mammalian ribozymes. We analyzed the folding of genomic HDV ribozymes by free energy minimization and found that variants with purine–pyrimidine combinations at the cleavage site are predicted to form native structures while pyrimidine–purine combinations misfold, consistent with earlier kinetic data and sequence comparisons. To test whether the cleavage site base pair contributes to catalysis, we characterized the pH and Mg2+-dependence of reaction kinetics of fast-folding genomic HDV ribozymes with cleavage site base pair purine–pyrimidine combinations: G•U, A-U, G-C, and A+•C. Rates for these native-folding ribozymes displayed highly similar pH and Mg2+ concentration dependencies, with the exception of the A+•C ribozyme...

tRNase Z Catalysis and Conserved Residues on the Carboxy Side of the His Cluster

Karkashon, Shay; Hopkinson, Angela; Levinger, Louis
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3′ end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the β-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II, and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reducesefficiency by ~1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in a proton transfer at the final stage of reaction...

Routes to covalent catalysis by reactive selection for nascent protein nucleophiles

Reshetnyak, Andrey V.; Armentano, Maria Francesca; Ponomarenko, Natalia A.; Vizzuso, Domenica; Durova, Oxana M.; Ziganshin, Rustam; Serebryakova, Marina; Govorun, Vadim; Gololobov, Gennady; Morse III, Herbert C.; Friboulet, Alain; Makker, Sudesh P.; Gabib
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) in order to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and non-covalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase*

Koszelak-Rosenblum, Mary; Krol, Adam C.; Simmons, Danielle M.; Goulah, Christopher C.; Wroblewski, Liliana; Malkowski, Michael G.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 05/09/2008 Português
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Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid α-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in Km, whereas no substantial change in Km was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids...

Glycosyltransferase Mechanisms: Impact of a 5-Fluoro Substituent in Acceptor and Donor Substrates on Catalysis†

Hartman, Matthew C.T.; Jiang, Songmin; Rush, Jeffrey S.; Waechter, Charles J.; Coward, James K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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In glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogs on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and β–N-acetylglucosaminyl-β-1,4 galactosyltransferase (GalT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc β-octyl glycoside acceptor; however...

Enzymatic Catalysis of Proton Transfer at Carbon: Activation of Triosephosphate Isomerase by Phosphite Dianion†

Amyes, Tina L.; Richard, John P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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More than 80% of the rate acceleration for enzymatic catalysis of the aldose-ketose isomerization of (R)-glyceraldehyde 3-phosphate (GAP) by triosephosphate isomerase (TIM) can be attributed to the 3′-phosphodianion group of GAP. We examine here the necessity of the covalent connection between the phosphodianion and triose sugar portions of the substrate by “carving up” GAP into the minimal neutral two-carbon sugar glycolaldehyde and phosphite dianion pieces. This “two-part substrate” preserves both the α-hydroxycarbonyl and oxydianion portions of GAP. TIM catalyzes proton transfer from glycolaldehyde in D2O, resulting in deuterium incorporation that can be monitored by 1H NMR spectroscopy, with kcat/Km = 0.26 M-1 s-1. Exogenous phosphite dianion results in a very large increase in the observed second-order rate constant (kcat/Km)obsd for turnover of glycolaldehyde, and the dependence of (kcat/Km)obsd on [HPO32-] exhibits saturation. The data give kcat/Km = 185 M-1s-1 for turnover of glycolaldehyde by TIM that is saturated with phosphite dianion, so that the separate binding of phosphite dianion to TIM results in a 700-fold acceleration of proton transfer from carbon. The binding of phosphite dianion to the free enzyme (Kd = 38 mM) is 700-fold weaker than its binding to the fleeting complex of TIM with the altered substrate in the transition state (Kd‡ = 53 μM); the total intrinsic binding energy of phosphite dianion in the transition state is 5.8 kcal/mol. We propose a physical model for catalysis by TIM in which the intrinsic binding energy of the substrate phosphodianion group is utilized to drive closing of the “mobile loop” and a protein conformational change that leads to formation of an active site environment that is optimally organized for stabilization of the transition state for proton transfer from α-carbonyl carbon.

The role of protein dynamics in thymidylate synthase catalysis: Variants of conserved dUMP-binding Tyr-261†

Newby, Zachary; Lee, Tom T.; Morse, Richard J.; Liu, Lu; Liu, Yaoquan; Venkatraman, Prasanna; Santi, Daniel V.; Finer-Moore, Janet S.; Stroud, Robert M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 20/06/2006 Português
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The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2′-deoxyuridine 5′-monophosphate (dUMP) to 2′-deoxythymidine 5′-monophosphate. Using kinetic and x-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3′-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Kms for both substrate and the cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of E. coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.

Natural Products as Inspiration for the Development of Asymmetric Catalysis

Mohr, Justin T.; Krout, Michael R.; Stoltz, Brian M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 18/09/2008 Português
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Biologically active natural products often contain particularly challenging structural features and functionalities. Perhaps foremost among these difficulties are issues of stereochemistry. A useful strategy for synthesizing these molecules is to devise novel methods of bond-formation that provide new opportunities for enantioselective catalysis. In using this tactic, target structures define the problems to be solved and ultimately drive development of catalysis forward. New enantioselective methods discovered in the context of these total synthesis efforts then contribute to a greater understanding of fundamental bond construction and lead to valuable synthetic technologies useful for a variety of other applications.

Triple-helix structure in telomerase RNA contributes to catalysis

Qiao, Feng; Cech, Thomas R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Telomerase is responsible for replication of the ends of linear chromosomes in most eukaryotes. Its intrinsic RNA subunit provides the template for synthesis of telomeric DNA by the reverse-transcriptase (TERT) subunit and tethers other proteins into the ribonucleoprotein (RNP) complex. We report that a phylogenetically conserved triple helix within a pseudoknot structure of this RNA contributes to telomerase activity but not by binding the TERT protein. Instead, 2′-OH groups protruding from the triple helix participate in both yeast and human telomerase catalysis; they may orient the primer-template relative to the active site in a manner analogous to group I ribozymes. The role of RNA in telomerase catalysis may have been acquired relatively recently or, alternatively, telomerase may be a molecular fossil representing an evolutionary link between RNA enzymes and RNP enzymes.

Ribozyme Catalysis Revisited: Is Water Involved?

Walter, Nils G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 28/12/2007 Português
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Enzymatic catalysis by RNA was discovered 25 years ago, yet mechanistic insights are emerging only slowly. Thought to be metalloenzymes at first, some ribozymes proved more versatile than anticipated when shown to utilize their own functional groups for catalysis. Recent evidence suggests that some may also judiciously place structural water molecules to shuttle protons in acid-base catalyzed reactions.

Oxidative Palladium(II) Catalysis: A Highly Efficient and Chemoselective Cross-Coupling Method for Carbon-Carbon Bond Formation under Base-Free and Nitrogenous-Ligand Conditions

Yoo, Kyung Soo; Yoon, Cheol Hwan; Jung, Kyung Woon
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 20/12/2006 Português
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We report herein the development of a general and mild protocol of oxygen promoted Pd(II) catalysis resulting in the selective cross-couplings of alkenyl- and arylboron compounds with various olefins. Unlike most cross-coupling reactions, this new methodology works well even in the absence of bases, consequently averting undesired homo-couplings. Nitrogen-based ligands including dimethyl-phenanathroline enhance reactivities and offer a highly efficient and stereoselective methodology to overcome challenging substrate limitations. For instance, oxidative palladium(II) catalysis is effective with highly substituted alkenes and cyclic alkenes, which are known to be incompatible with other known catalytic conditions. Most examined reactions progressed smoothly to completion at low temperatures and in short times. These interesting results provide mechanistic insights and utilities for a new paradigm of palladium catalytic cycles without bases.

Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds*S⃞

Taguchi, Hiroaki; Planque, Stephanie; Sapparapu, Gopal; Boivin, Stephane; Hara, Mariko; Nishiyama, Yasuhiro; Paul, Sudhir
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 26/12/2008 Português
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Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid β (Aβ)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Aβ-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Aβ analog identified rare IgVs that hydrolyzed Aβ mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t′). The IgVs hydrolyzed Aβ at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Aβ hydrolysis, suggesting catalysis by the unpaired VL domain.Ångstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity...

Solvent Structure and Hammerhead Ribozyme Catalysis

Martick, Monika; Lee, Tai-Sung; York, Darrin M.; Scott, William G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/2008 Português
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Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 Å structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn2+ is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2′-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.

Explaining an Unusually Fast Parasitic Enzyme: Folate Tail-Binding Residues Dictate Substrate Positioning and Catalysis in Cryptosporidium hominis Thymidylate Synthase†,‡

Martucci, W. Edward; Vargo, Melissa A.; Anderson, Karen S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The essential enzyme TS-DHFR from Cryptosporidium hominis undergoes an unusually rapid rate of catalysis at the conserved TS domain, facilitated by two nonconserved residues, Ala287 and Ser290, in the folate tail-binding region. Mutation of these two residues to their conserved counterparts drastically affects multiple steps of the TS catalytic cycle. We have determined the crystal structures of all three mutants (A287F, S290G, and A287F/S290G) in complex with active site ligands dUMP and CB3717. The structural data show two effects of the mutations: an increased distance between the ligands in the active site and increased flexibility of the folate ligand in the partially open enzyme state that precedes conformational change to the active catalytic state. The latter effect is able to be rescued by the mutants containing the A287F mutation. In addition, the conserved water network of TS is altered in each of the mutants. The structural results point to a role of the folate tail-binding residues in closely positioning ChTS ligands and restricting ligand flexibility in the partially open state to allow for a rapid transition to the active closed state and enhanced rate of catalysis. These results provide an explanation on how folate tail-binding residues at one end of the active site affect long-range interactions throughout the TS active site and validate these residues as targets for species-specific drug design.

Molecular Basis for Enzymatic Sulfite Oxidation: HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY*S⃞

Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei V.; Enemark, John H.; Kappler, Ulrike
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 23/01/2009 Português
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Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2–3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (∼60 and 200 s–1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site...

Covalent Catalysis by Pyridoxal: Evaluation of the Effect of the Cofactor on the Carbon Acidity of Glycine

Toth, Krisztina; Richard, John P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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First-order rate constants for deprotonation of the α-imino carbon of the adduct between 5′-deoxypyridoxal (1) and glycine were determined as the rate constants for Claisen-type addition of glycine to 1 where deprotonation is rate determining for product formation. There is no significant deprotonation at pH 7.1 of the form of the 1-glycine iminium ion with the pyridine nitrogen in the basic form. The value of kHO for hydroxide ion-catalyzed deprotonation of the α-imino carbon increases from 7.5 × 102 to 3.8 × 105 to 3.0 × 107 M-1 s-1, respectively, with protonation of the pyridine nitrogen, the phenoxide oxyanion and the carboxylate anion of the 1-glycine iminium ion. There is a corresponding decrease in the pKas for deprotonation of the α-imino carbon from 17 to 11 to 6. It is proposed that enzymes selectively bind and catalyze the reaction of the iminium ion with pKa = 17. A comparison of kB = 1.7 × 10-3 s-1 for deprotonation of the α-imino carbon of this cofactor-glycine adduct (pKa = 17 by HPO42- with kcat/Km = 4 × 105M-1 s-1 for catalysis of amino-acid racemization by alanine racemase shows that the enzyme causes a ca 2 × 108-fold acceleration of the rate of deprotonation the α-imino carbon. This corresponds to about one-half of the burden borne by alanine racemase in catalysis of deprotonation of alanine.

Defining the Potassium Binding Region in an Apple Terpene Synthase*S⃞

Green, Sol; Squire, Christopher J.; Nieuwenhuizen, Niels J.; Baker, Edward N.; Laing, William
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 27/03/2009 Português
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Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M2+) metal ion-assisted electrophilic attack. In addition to M2+, monovalent cations (M+) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating substrate binding or catalysis. An apple α-farnesene synthase (MdAFS1), which has a dependence upon potassium (K+), was used to identify active site regions that may be important for M+ binding. Protein homology modeling revealed a surface-exposed loop (H-αl loop) in MdAFS1 that fulfilled the necessary requirements for a K+ binding region. Site-directed mutagenesis analysis of specific residues within this loop then revealed their crucial importance to this K+ response and strongly implicated specific residues in direct K+ binding. The role of the H-αl loop in terpene synthase K+ coordination was confirmed in a Conifer pinene synthase also using site-directed mutagenesis. These findings provide the first direct evidence for a specific M+ binding region in two functionally and phylogenetically divergent terpene synthases. They also provide a basis for understanding K+ activation in other terpene synthases and establish a new role for the H-αl loop region in terpene synthase catalysis.

Substrate Recognition of Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase from Prochlorococcus marinus*

Bröcker, Markus J.; Wätzlich, Denise; Uliczka, Frank; Virus, Simone; Saggu, Miguel; Lendzian, Friedhelm; Scheer, Hugo; Rüdiger, Wolfhart; Moser, Jürgen; Jahn, Dieter
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 31/10/2008 Português
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Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL2 and a heterotetrameric protein complex (ChlNB)2. The ChlL2 dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB)2 complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB)2. For the analysis of DPOR substrate recognition...

Theoretical Study of the Catalysis of Cyanohydrin Formation by the Cyclic Dipeptide Catalyst cyclo[(S)-His-(S)-Phe]

Schoenebeck, Franziska; Houk, K. N.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 20/02/2009 Português
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Dipeptide cyclo[(S)-His-(S)-Phe] 1, first applied by Inoue et al. in 1981, catalyzes the hydrocyanation of aromatic aldehydes very efficiently. Enantioselective autoinduction has also been reported for the process. We have employed QM (Density Functional Theory and MP2), Molecular Mechanics (MM) and Molecular Dynamics (MD) methods to (i) derive a mechanistic picture for catalysis and (ii) reveal the origin of stereochemistry and autoinduction. A dimer is proposed to be the catalytic species, in which one imidazole group is essential for the delivery of the nucleophile and the second imidazole group acts as an acid, accompanied with π-interaction for most favorable substrate binding. H-bonding via hydroxy groups is crucial for catalysis also. MD studies indicate stability of the dimer only in non-polar media, which is consistent with the need of the experimental (heterogeneous) reaction conditions to achieve high enantioselectivities. DFT and MP2 results suggest the incorporation of the product cyanohydrin via extended edge-to-face π-interaction over three aromatic units. Transition states derived from this model are in good agreement with experimental findings and enantioselectivities.