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Roles of static and dynamic domains in stability and catalysis of adenylate kinase

Bae, Euiyoung; Phillips, George N.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Protein dynamics, including conformational switching, are recognized to be crucial for the function of many systems. These motions are more challenging to study than simple static structures. Here, we present evidence suggesting that in the enzyme adenylate kinase large “hinge bending” motions closely related to catalysis are regulated by intrinsic properties of the moving domains and not by their hinges, by anchoring domains, or by remote allosteric-like regions. From a pair of highly homologous mesophilic and thermophilic adenylate kinases, we generated a series of chimeric enzymes using a previously undescribed method with synthetic genes. Subsequent analysis of the chimeras has revealed unexpected spatial separation of stability and activity control. Our results highlight specific contributions of dynamics to catalysis in adenylate kinase. Furthermore, the overall strategy and the specific mutagenesis method used in this study can be generally applied.

Clusters, surfaces, and catalysis

Somorjai, Gabor A.; Contreras, Anthony M.; Montano, Max; Rioux, Robert M.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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The surface science of heterogeneous metal catalysis uses model systems ranging from single crystals to monodispersed nanoparticles in the 1–10 nm range. Molecular studies reveal that bond activation (C–H, H–H, C–C, CO) occurs at 300 K or below as the active metal sites simultaneously restructure. The strongly adsorbed molecules must be mobile to free up these sites for continued turnover of reaction. Oxide–metal interfaces are also active for catalytic turnover. Examples using C–H and CO activation are described to demonstrate these properties. Future directions include synthesis, characterization, and reaction studies with 2D and 3D monodispersed metal nanoclusters to obtain 100% selectivity in multipath reactions. Investigations of the unique structural, dynamic, and electronic properties of nanoparticles are likely to have major impact in surface technologies. The fields of heterogeneous, enzyme, and homogeneous catalysis are likely to merge for the benefit of all three.

Evidence that substrate-specific effects of C5 protein lead to uniformity in binding and catalysis by RNase P

Sun, Lei; Campbell, Frank E; Zahler, Nathan H; Harris, Michael E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The ribonucleoprotein enzyme RNase P processes all pre-tRNAs, yet some substrates apparently lack consensus elements for recognition. Here, we compare binding affinities and cleavage rates of Escherichia coli pre-tRNAs that exhibit the largest variation from consensus recognition sequences. These results reveal that the affinities of both consensus and nonconsensus substrates for the RNase P holoenzyme are essentially uniform. Comparative analyses of pre-tRNA and tRNA binding to the RNase P holoenzyme and P RNA alone reveal differential contributions of the protein subunit to 5′ leader and tRNA affinity. Additionally, these studies reveal that uniform binding results from variations in the energetic contribution of the 5′ leader, which serve to compensate for weaker tRNA interactions. Furthermore, kinetic analyses reveal uniformity in the rates of substrate cleavage that result from dramatic (>900-fold) contributions of the protein subunit to catalysis for some nonconsensus pre-tRNAs. Together, these data suggest that an important biological function of RNase P protein is to offset differences in pre-tRNA structure such that binding and catalysis are uniform.

Free-energy profiles for catalysis by dual-specificity phosphatases

Arantes, Guilherme M.
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
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PTPs (protein tyrosine phosphatases) are fundamental enzymes for cell signalling and have been linked to the pathogenesis of several diseases, including cancer. Hence, PTPs are potential drug targets and inhibitors have been designed as possible therapeutic agents for Type II diabetes and obesity. However, a complete understanding of the detailed catalytic mechanism in PTPs is still lacking. Free-energy profiles, obtained by computer simulations of catalysis by a dual-specificity PTP, are shown in the present study and are used to shed light on the catalytic mechanism. A highly accurate hybrid potential of quantum mechanics/molecular mechanics calibrated specifically for PTP reactions was used. Reactions of alkyl and aryl substrates, with different protonation states and PTP active-site mutations, were simulated. Calculated reaction barriers agree well with experimental rate measurements. Results show the PTP substrate reacts as a bi-anion, with an ionized nucleophile. This protonation state has been a matter of debate in the literature. The inactivity of Cys→Ser active-site mutants is also not fully understood. It is shown that mutants are inactive because the serine nucleophile is protonated. Results also clarify the interpretation of experimental data...

Identification of Tyr413 as an Active Site Residue in the Flavoprotein Tryptophan 2-Monooxygenase and Analysis of Its Contribution to Catalysis†

Sobrado, Pablo; Fitzpatrick, Paul F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 02/12/2003 Português
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The flavoenzyme tryptophan 2-monooxygenase catalyzes the oxidation of tryptophan to indoleacetamide, carbon dioxide, and water. The enzyme is a homologue of L-amino acid oxidase. In the structure of L-amino acid oxidase complexed with aminobenzoate, Tyr372 hydrogen bonds with the carboxylate of the inhibitor in the active site. All 10 conserved tyrosine residues in tryptophan 2-monooxygenase were mutated to phenylalanine; steady state kinetic characterization of the purified proteins identified Tyr413 as the residue homologous to Tyr372 of L-amino acid oxidase. Y413F and Y413A tryptophan 2-monooxygenase were characterized more completely with tryptophan as the substrate to probe the contribution of this residue to catalysis. Mutation of Tyr413 to phenylalanine results in a decrease in the value of the first-order rate constant for reduction of 35-fold and a decrease in the rate constant for oxidation of 11-fold. Mutation to alanine decreases the rate constant for reduction by 200-fold and that for oxidation by 33-fold. Both mutations increase the Kd value for tryptophan and the Ki values for the competitive inhibitors indoleacetamide and indole pyruvate by 5–10-fold. Both mutations convert the enzyme to an oxidase, in that the products of the catalytic reactions of both are indolepyruvate and hydrogen peroxide. The V/Ktrp −pH profiles for the Tyr413 mutant enzymes no longer show the pKa value of 9.9 seen in that for the wild-type enzyme...

Protein motions during catalysis by dihydrofolate reductases

Allemann, Rudolf K; Evans, Rhiannon M; Tey, Lai-hock; Maglia, Giovanni; Pang, Jiayun; Rodriguez, Robert; Shrimpton, Paul J; Swanwick, Richard S
Fonte: The Royal Society Publicador: The Royal Society
Tipo: Artigo de Revista Científica
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Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima. We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.

Transition state theory can be used in studies of enzyme catalysis: lessons from simulations of tunnelling and dynamical effects in lipoxygenase and other systems

Olsson, Mats H.M; Mavri, Janez; Warshel, Arieh
Fonte: The Royal Society Publicador: The Royal Society
Tipo: Artigo de Revista Científica
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The idea that enzyme catalysis involves special factors such as coherent fluctuations, quantum mechanical tunnelling and non-equilibrium solvation (NES) effects has gained popularity in recent years. It has also been suggested that transition state theory (TST) cannot be used in studies of enzyme catalysis. The present work uses reliable state of the art simulation approaches to examine the above ideas. We start by demonstrating that we are able to simulate any of the present catalytic proposals using the empirical valence bond (EVB) potential energy surfaces, the dispersed polaron model and the quantized classical path (QCP) approach, as well as the approximate vibronic method. These approaches do not treat the catalytic effects by phenomenological treatments and thus can be considered as first principles approaches (at least their ability to compare enzymatic reaction to the corresponding solution reactions). This work will consider the lipoxygenase reaction, and to lesser extent other enzymes, for specific demonstration. It will be pointed out that our study of the lipoxygenase reaction reproduces the very large observed isotope effect and the observed rate constant while obtaining no catalytic contribution from nuclear quantum mechanical (NQM) effects. Furthermore...

Structural model of F1-ATPase and the implications for rotary catalysis.

Leslie, A G; Walker, J E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 29/04/2000 Português
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The crystal structure of bovine mitochondrial F1-ATPase is described. Several features of the structure are consistent with the binding change mechanism of catalysis, in which binding of substrates induces conformational changes that result in a high degree of cooperativity between the three catalytic sites. Furthermore, the structure also suggests that catalysis is accompanied by a physical rotation of the centrally placed gamma-subunit relative to the approximately spherical alpha3beta3 subassembly.

Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay

Diamond, Tracy L.; Bushman, Frederic D.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5′ hydroxyl groups as well as 3′ hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions...

The 1′,4′-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes

Nemeria, Natalia; Chakraborty, Sumit; Baykal, Ahmet; Korotchkina, Lioubov G.; Patel, Mulchand S.; Jordan, Frank
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4′-aminopyrimidine aromatic rings, but only recently has participation of the 4′-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4′-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the E1 component of human pyruvate dehydrogenase complex, the E1 subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1′,4′-iminopyrimidine and the 4′-aminopyrimidine tautomers coexist on the E1 component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate...

Glutamate-64, a newly identified residue of the functionally conserved electron-sharing network contributes to catalysis and structural integrity of glutathione transferases

Winayanuwattikun, Pakorn; Ketterman, Albert J.
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
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In Anopheles dirus glutathione transferase D3-3, position 64 is occupied by a functionally conserved glutamate residue, which interacts directly with the γ-glutamate moiety of GSH (glutathione) as part of an electron-sharing network present in all soluble GSTs (glutathione transferases). Primary sequence alignment of all GST classes suggests that Glu64 is one of a few residues that is functionally conserved in the GST superfamily. Available crystal structures as well as consideration of the property of the equivalent residue at position 64, acidic or polar, suggest that the GST electron-sharing motif can be divided into two types. Electrostatic interaction between the GSH glutamyl and carboxylic Glu64, as well as with Arg66 and Asp100, was observed to extend the electron-sharing motif identified previously. Glu64 contributes to the catalytic function of this motif and the ‘base-assisted deprotonation’ that are essential for GSH ionization during catalysis. Moreover, this residue also appears to affect multiple steps in the enzyme catalytic strategy, including binding of GSH, nucleophilic attack by thiolate at the electrophilic centre and product formation, probably through active-site packing effects. Replacement with non-functionally-conserved amino acids alters initial packing or folding by favouring aggregation during heterologous expression. Thermodynamic and reactivation in vitro analysis indicated that Glu64 also contributes to the initial folding pathway and overall structural stability. Therefore Glu64 also appears to impact upon catalysis through roles in both initial folding and structural maintenance.

DEOXYHYPUSINE HYDROXYLASE IS A Fe(II)-DEPENDENT, HEAT-REPEAT ENZYME: IDENTIFICATION OF AMINO ACID RESIDUES CRITICAL FOR Fe(II) BINDING AND CATALYSIS

Kim, Yeon Sook; Kang, Kee Ryeon; Wolff, Edith C.; Bell, Jessica K.; McPhie, Peter; Park, Myung Hee
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) comprised of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron per mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by addition of Fe(II) to the apoenzyme, but not by other metals including Cd, Co, Cr, Cu, Mg, Mn, Ni and Zn. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including His56A, His89A, Glu90A, His207A, His240A and Glu241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore...

A guanine nucleobase important for catalysis by the VS ribozyme

Wilson, Timothy J; McLeod, Aileen C; Lilley, David M J
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
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A guanine (G638) within the substrate loop of the VS ribozyme plays a critical role in the cleavage reaction. Replacement by any other nucleotide results in severe impairment of cleavage, yet folding of the substrate is not perturbed, and the variant substrates bind the ribozyme with similar affinity, acting as competitive inhibitors. Functional group substitution shows that the imino proton on the N1 is critical, suggesting a possible role in general acid–base catalysis, and this in accord with the pH dependence of the reaction rate for the natural and modified substrates. We propose a chemical mechanism for the ribozyme that involves general acid–base catalysis by the combination of the nucleobases of guanine 638 and adenine 756. This is closely similar to the probable mechanism of the hairpin ribozyme, and the active site arrangements for the two ribozymes appear topologically equivalent. This has probably arisen by convergent evolution.

Acid–base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237→Gln mutant enzymes

Schwarz, Alexandra; Brecker, Lothar; Nidetzky, Bernd
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
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The role of acid–base catalysis in the two-step enzymatic mechanism of α-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237→Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 105-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding α-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed α-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/Km, the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pKa values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pKa≈5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pKa≈6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid–base of sucrose phosphorylase. They support a mechanism in which the pKa of Glu237 cycles between ≈7.2 in free enzyme and ≈5.8 in glucosyl enzyme intermediate...

An interpretation of fluctuations in enzyme catalysis rate, spectral diffusion, and radiative component of lifetimes in terms of electric field fluctuations

Prakash, Meher K.; Marcus, R. A.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Time-dependent fluctuations in the catalysis rate (δk(t)) observed in single-enzyme experiments were found in a particular study to have an autocorrelation function decaying on the same time scale as that of spectral diffusion δω0(t). To interpret this similarity, the present analysis focuses on a factor in enzyme catalysis, the local electrostatic interaction energy (E) at the active site and its effect on the activation free energy barrier. We consider the slow fluctuations of the electrostatic interaction energy (δE(t)) as a contributor to δk(t) and relate the latter to δω0(t). The resulting relation between δk(t) and δω0(t) is a dynamic analog of the solvatochromism used in interpreting solvent effects on organic reaction rates. The effect of the postulated δE(t) on fluctuations in the radiative component (δγr−1(t)) of the fluorescence decay of chromophores in proteins also is examined, and a relation between δγr−1(t) and δω0(t) is obtained. Experimental tests will determine whether the correlation functions for δk(t), δω0(t), and δγr−1 are indeed similar for any enzyme. Measurements of dielectric dispersion, ε(ω), for the enzyme discussed elsewhere will provide further insight into the correlation function for δE(t). They also will determine whether fluctuations in the nonradiative component γnr−1 of the lifetime decay has a different origin...

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Goward, C. R.; Nicholls, D. J.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /10/1994 Português
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Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.

Structural Basis for Catalysis by Onconase

Lee, J. Eugene; Bae, Euiyoung; Bingman, Craig A.; Phillips, George N.; Raines, Ronald T.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Onconase (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC·nucleic acid complexes: a T89N/E91A ONC·5′-AMP complex at 1.65 Å resolution and a wild-type ONC·d(AUGA) complex at 1.90 Å resolution. The latter structure and site-directed mutagenesis was used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH–kcat/KM profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 102-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known kcat/KM value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC·d(AUGA) complex was also used to probe other aspects of catalysis. For example...

Identification of the Lateral Interaction Surfaces of Human Histocompatibility Leukocyte Antigen (HLA)-DM with HLA-DR1 by Formation of Tethered Complexes That Present Enhanced HLA-DM Catalysis

Stratikos, Efstratios; Mosyak, Lidia; Zaller, Dennis M.; Wiley, Don C.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 15/07/2002 Português
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Human histocompatibility leukocyte antigen (HLA)-DM is a major histocompatibility complex (MHC)-like protein that catalyzes exchange of antigenic peptides from MHC class II molecules. To investigate the molecular details of this catalysis we created four covalent complexes between HLA-DM and the MHC class II allele DR1. We introduced a disulfide bond between the naturally occurring cysteine β46 on HLA-DM and an engineered cysteine on the end of a linker attached to either the NH2- or the COOH terminus of an antigenic peptide that is tightly bound on DR1. We find that when DM is attached to the NH2 terminus of the peptide, it can, for all linker lengths tested, catalyze exchange of the peptide with a half-life a few minutes (compared with uncatalyzed t1/2 > 100 h). This rate, which is several orders of magnitude greater than the one we obtain in solution assays using micromolar concentrations of HLA-DM, is dominated by a concentration independent factor, indicating an intramolecular catalytic interaction within the complex. A similar complex formed at the COOH terminus of the peptide shows no sign of DM-specific intramolecular catalysis. Restrictions on the possible interaction sites imposed by the length of the linkers indicate that the face of DR1 that accommodates the NH2 terminus of the antigenic peptide interacts with the lateral face of HLA-DM that contains cysteine β46.

Modular organization of FDH: Exploring the basis of hydrolase catalysis

Reuland, Steven N.; Vlasov, Alexander P.; Krupenko, Sergey A.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /05/2006 Português
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An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an ~100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.

The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

Kleifeld, Oded; Shi, Shu Ping; Zarivach, Raz; Eisenstein, Miriam; Sagi, Irit
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /03/2003 Português
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Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore...