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Energetic contribution of non-essential 5′ sequence to catalysis in a hepatitis delta virus ribozyme

Shih, I.-hung; Been, Michael D.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 03/09/2001 Português
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Hepatitis delta virus (HDV) ribozymes employ multiple catalytic strategies to achieve overall rate enhancement of RNA cleavage. These strategies include general acid–base catalysis by a cytosine side chain and involvement of divalent metal ions. Here we used a trans-acting form of the antigenomic ribozyme to examine the contribution of the 5′ sequence in the substrate to HDV ribozyme catalysis. The cleavage rate constants increased for substrates with 5′ sequence alterations that reduced ground-state binding to the ribozyme. Quantitatively, a plot of activation free energy of chemical conversion versus Gibb’s free energy of substrate binding revealed a linear relationship with a slope of –1. This relationship is consistent with a model in which components of the substrate immediately 5′ to the cleavage site in the HDV ribozyme–substrate complex destabilize ground-state binding. The intrinsic binding energy derived from the ground-state destabilization could contribute up to 2 kcal/mol toward the total 8.5 kcal/mol reduction in activation free energy for RNA cleavage catalyzed by the HDV ribozyme.

The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis.

Lykke-Andersen, J; Christiansen, J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/12/1998 Português
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DNA and RNA polymerases use divalent metal ions for catalysis. Crystal structures of several polymerases reveal that two acidic residues are involved in coordinating two metal ions at the catalytic centre. Bacteriophage RNA polymerases contain a highly conserved C-terminus with the carboxylate positioned near the active site. We examined whether theC-terminal carboxy group of T7 RNA polymerase is important for magnesium ion-dependent catalysis. Introduction of a methyl ester or decarboxylation of the C-terminal carboxy group was achieved with an intein-based protein expression system and an elongation rate assay was developed to test the effects of the modifications. The results show that enzymes with a modified C-terminal carboxy group exhibit a magnesium ion-dependent decrease in catalytic activity.

Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease

Tock, Mark R.; Frary, Elaine; Sayers, Jon R.; Grasby, Jane A.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 03/03/2003 Português
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On the basis of structural work, metal ions are proposed to play a catalytic role in reactions mediated by many phosphoryl transfer enzymes. To gain dynamic support for such mechanisms, the role of metal ion cofactors in phosphate diester hydrolysis catalysed by a flap endonuclease has been studied. The pH maximal rate profiles were measured in the presence of various metal ion cofactors; in each case, a single ionic form of the enzyme/cofactor accounts for the pH dependence. The kinetic pKas display good correlation with the acidity of the corresponding hexahydrated metal ions, which strongly suggests a role for metal-bound hydroxide, or its equivalent ionic species, in the reaction. Comparing rates of reaction in the pH-independent regions, a small negative βnuc value is observed. This suggests that expected trends in the nucleophilicity of the various metal-bound hydroxides are balanced by a second form of metal ion catalysis that is related to the acidity of the hexahydrated metal ion. This is likely to be either electrophilic catalysis or leaving group activation.

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme of Salmonella enterica Are Essential for Catalysis

Grimek, T. L.; Holden, H.; Rayment, I.; Escalante-Semerena, J. C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2003 Português
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The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg2+ ions; equimolar concentrations of Mn2+ ions were a poor substitute for Mg2+ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The Km of PrpB for 2-MIC was measured at 19 μM, with a kcat of 105 s−1. Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58...

An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis.

Herschlag, D; Khosla, M; Tsuchihashi, Z; Karpel, R L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/1994 Português
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We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions.

Uptake of oleate from albumin solutions by rat liver. Failure to detect catalysis of the dissociation of oleate from albumin by an albumin receptor.

Weisiger, R A; Ma, W L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1987 Português
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The hepatic removal of albumin-bound substances from plasma requires that they dissociate from albumin. Using indirect methods, we and others have proposed that dissociation may be catalyzed by interaction of albumin with the liver cell surface. This study looked for direct evidence of catalysis by comparing the rate of dissociation of oleate from albumin in vitro with the rate observed within the sinusoids of perfused rat liver. No evidence for catalysis was found. The rate of hepatic oleate removal from dilute albumin solutions did not exceed but instead closely paralleled the rate predicted from the in vitro dissociation rate constant (0.14s-1). These results suggest that under some conditions the liver can remove unbound material from the sinusoids faster than it can be replenished by dissociation from albumin, resulting in dissociation-limited removal. However, dissociation of oleate does not appear to be catalyzed by the liver.

Catalysis by acetylcholinesterase: evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis.

Rosenberry, T L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1975 Português
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Inferences about the catalytic mechanism of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are frequently made on the basis of a presumed analogy with chymotrypsin, EC 3.4.21.1. Although both enzymes are serine hydrolases, several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, kcat/Kapp. While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysis, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of kcat/Kapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves nonlinear inhibition by [H+]. Deuterium oxide isotope effects for kcat/Kapp range from 1.1 for acetylcholine to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate.

Piezoelectric Theory of Enzymic Catalysis as Inferred from the Electromechanochemical Principles of Bioenergetics

Caserta, Giuseppe; Cervigni, Tommaso
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1974 Português
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Starting from the electromechanochemical principles of bioenergetics formulated by Green and Ji, a theory is proposed which describes enzymic catalysis in terms of piezoelectricity in semiconductors. The choice of this particular physical effect for describing catalytic processes is founded on the following experimental observations: most of the amino-acid residues of enzymes, as well as a large number of other biologically important molecules, exhibit piezoelectric resonances; besides, enzymes can behave like semiconductors. In the proposed theory the catalysis is assumed to be accomplished by means of three fundamental processes: (a) the lowering of the substrate-product energy barrier; (b) the electron-induced selective amplification of the low-frequency vibrational waves present in thermal background in the enzyme structure; and (c) the channeling into the substrate of the energy associated with the amplified waves and utilization of this energy for generating electrical or mechanical fields inside a susceptible region of the substrate. A mathematical description of the theory is outlined, and a rough estimate of some quantities involved in the process of wave amplification is also reported.

Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance

Rueda, David; Bokinsky, Gregory; Rhodes, Maria M.; Rust, Michael J.; Zhuang, Xiaowei; Walter, Nils G.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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The hairpin ribozyme is a minimalist paradigm for studying RNA folding and function. In this enzyme, two domains dock by induced fit to form a catalytic core that mediates a specific backbone cleavage reaction. Here, we have fully dissected its reversible reaction pathway, which comprises two structural transitions (docking/undocking) and a chemistry step (cleavage/ligation), by applying a combination of single-molecule fluorescence resonance energy transfer (FRET) assays, ensemble cleavage assays, and kinetic simulations. This has allowed us to quantify the effects that modifications of essential functional groups remote from the site of catalysis have on the individual rate constants. We find that all ribozyme variants show similar fractionations into effectively noninterchanging molecule subpopulations of distinct undocking rate constants. This leads to heterogeneous cleavage activity as commonly observed for RNA enzymes. A modification at the domain junction additionally leads to heterogeneous docking. Surprisingly, most modifications not only affect docking/undocking but also significantly impact the internal chemistry rate constants over a substantial distance from the site of catalysis. We propose that a network of coupled molecular motions connects distant parts of the RNA with its reaction site...

An enzymatic molten globule: Efficient coupling of folding and catalysis

Vamvaca, Katherina; Vögeli, Beat; Kast, Peter; Pervushin, Konstantin; Hilvert, Donald
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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A highly active, monomeric chorismate mutase, obtained by topological redesign of a dimeric helical bundle enzyme from Methanococcus jannaschii, was investigated by NMR and various other biochemical techniques, including H/D exchange. Although structural disorder is generally considered to be incompatible with efficient catalysis, the monomer, unlike its natural counterpart, unexpectedly possesses all of the characteristics of a molten globule. Global conformational ordering, observed upon binding of a transition state analog, indicates that folding can be coupled to catalysis with minimal energetic penalty. These results support the suggestion that many modern enzymes might have evolved from molten globule precursors. Insofar as their structural plasticity confers relaxed substrate specificity and/or catalytic promiscuity, molten globules may also be attractive starting points for the evolution of new catalysts in the laboratory.

A Kinetic Characterization of Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis

Edmondson, Daryl L.; Badger, Murray R.; Andrews, T. John
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1990 Português
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The catalytic activity of ribulosebisphosphate carboxylase (Rubisco) declined as soon as catalysis was initiated by exposure to its substrate, d-ribulose-1,5-bisphosphate (ribulose-P2). The decline continued exponentially, with a half-time of approximately 7 minutes until, eventually, a steady state level of activity was reached which could be as low as 15% of the initial activity. The ratio of the steady state activity to the initial activity was lower at low CO2 concentration and at low pH. The inhibitors 6-phosphogluconate and H2O2 alleviated the inactivation, increasing the final/initial rate ratio and the half-time. Varying ribulose-P2 concentration in the range above that required to saturate catalysis did not affect the kinetics of inactivation. The affinities for CO2 and ribulose-P2 were unaffected by the inactivation. The decline in activity occurred with preparations of ribulose-P2 which contained no detectable d-xylulose-1,5-bisphosphate and also with ribulose-P2 which had been generated enzymatically immediately before use. Inclusion of an aldolase system for removing d-xylulose-1,5-bisphosphate also did not alter the inactivation process. The inactivated Rubisco did not recover after complete exhaustion of ribulose-P2. We conclude that the inactivation is not caused by readily-reversible binding of ribulose-P2 at a site different from the active site and that it is unlikely to be attributable to inhibitory contaminants in ribulose-P2 preparations.

Prolyl endopeptidase catalysis. A physical rather than a chemical step is rate-limiting.

Polgár, L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/05/1992 Português
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Prolyl endopeptidase represents a new family of serine proteases, and it has a mechanistic feature distinct from that of the enzymes of the extensively studied chymotrypsin and subtilisin families. The rate-determining step in the catalysis of serine proteases is a general base/acid-catalysed chemical step. For prolyl endopeptidase, however, the chemical step is not rate-limiting, as demonstrated by using substrates with different leaving groups. It is known that the acylation of chymotrypsin and subtilisin proceeds faster by several orders of magnitude with the activated nitrophenyl ester than with the corresponding amide substrates. In contrast, for the acylation of prolyl endopeptidase similar rate constants were obtained with nitrophenyl ester and several amide substrates. This result, combined with kinetic isotope studies [Polgár (1991) Eur. J. Biochem. 197, 441-447], offers strong evidence that a physical step, presumably a conformational change associated with substrate binding, is the rate-determining step in the prolyl endopeptidase catalysis.

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

Gul, Sheraz; Brown, Richard; May, Earl; Mazzulla, Marie; Smyth, Martin G.; Berry, Colin; Morby, Andrew; Powell, David J.
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
Português
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DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume)...

A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase.

Laing, W A; Christeller, J T
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/12/1976 Português
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Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses...

Mapping the active site of the Haemophilus influenzae methionyl-tRNA formyltransferase: residues important for catalysis and tRNA binding.

Newton, D T; Mangroo, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/04/1999 Português
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Formylation of the initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is an essential step in initiation of protein synthesis in eubacteria. Here, site-directed mutagenesis was used to identify active site residues of the Haemophilus influenzae MTF. Of the nine residues investigated, only Arg-41, Asn-107, His-109 and Asp-145 were important for the function of the H. influenzae MTF. Replacement of these residues with Ala resulted in a significant reduction in the efficiency of catalysis. Intrinsic fluorescence analysis indicated that this was not due to a defect in N10-formyltetrahydrofolate (fTHF) binding. The Asp-145 and Arg-41 mutations reduced the affinity of the enzyme for the initiator tRNA, whereas the Asn-107 and His-109 mutations affected catalysis but not tRNA binding. Replacement of Arg-41, His-109 and Asp-145 with functionally similar residues also affected the activity of the enzyme. The data suggest that Asn-107, His-109 and Asp-145 are catalytic residues, whereas Arg-41 is involved in tRNA recognition. In the Escherichia coli glycinamide ribonucleotide formyltransferase, which also uses fTHF as the formyl donor, Asn-106, His-108 and Asp-144 participate in the catalytic step. Together, these observations imply that this group of enzymes uses the same basic mechanism in formylating their substrates.

Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A.

Sánchez-Torres, Paloma; Visser, Jaap; Benen, Jacques A E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/02/2003 Português
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Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed beta-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. On the basis of the three-dimensional structures of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677-689] and the modelled enzyme-substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 8762-8769], Asp154, Arg176, Arg236 and Lys239 were mutagenized. Substituting Arg236 with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg176 and Lys239 severely affected catalysis. The Asp154-->Arg and Asp154-->Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg236, which is sandwiched between Arg176 and Lys239, would initiate the reaction upon enzyme-substrate interaction, through the abstraction of the proton at C5 of the galacturonopyranose ring. The positively charged residues Arg176 and Lys239 are responsible for lowering the p K a of Arg236. Arg176 and Lys239 are maintained in a charged state by interacting with Asp154 or bulk solvent respectively. The deprotonation of the Asp186-Asp221 pair was proposed to be responsible for a pH-driven conformational change of PL1A [Mayans...

Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis.

Pedersen, Anja K; Guo, Xiao-Ling; Møller, Karin B; Peters, Günther H; Andersen, Henrik S; Kastrup, Jette S; Mortensen, Steen B; Iversen, Lars F; Zhang, Zhong-Yin; Møller, Niels Peter H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/2004 Português
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Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.

Role of constraint in catalysis and high-affinity binding by proteins.

Vanselow, Donald G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/2002 Português
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Using a model for catalysis of a dynamic equilibrium, the role of constraint in catalysis is quantified. The intrinsic rigidity of proteins is shown to be insufficient to constrain the activated complexes of enzymes, irrespective of the mechanism. However, when minimization of the surface excess free energy of water surrounding a protein is considered, model proteins can be designed with regions of sufficient rigidity. Structures can be designed to focus surface tension or hydrophobic attraction as compressive stress. A monomeric structure has a limited ability to concentrate compressive stress and constrain activated complexes. Oligomeric or multidomain proteins, with domains surrounding a rigid core, have unlimited ability to concentrate stress, provided there are at least four domains. Under some circumstances, four is the optimum number, which could explain the frequency of tetrameric enzymes in nature. The minimum compressive stress in oligomers increases with the square of the radius. For tetramers of similar size to natural enzymes, this stress agrees reasonably well with that needed to constrain the activated complex. A similar principle applies to high affinity binding proteins. The models explain the trigonal pyramidal shape of fibroblast growth factor and provide a basis for interpretation of protein crystal structures.

Division of Labor within Human Immunodeficiency Virus Integrase Complexes: Determinants of Catalysis and Target DNA Capture†

Diamond, Tracy L.; Bushman, Frederic D.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2005 Português
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Following the completion of reverse transcription, the human immunodeficiency virus integrase (IN) enzyme covalently links the viral cDNA to a host cell chromosome. An IN multimer carries out this reaction, but the roles of individual monomers within the complex are mostly unknown. Here we analyzed the distribution of functions for target DNA capture and catalysis within the IN multimer. We used forced complementation between pairs of IN deletion derivatives in vitro as a tool for probing cis-trans relationships and analyzed amino acid substitutions affecting either catalysis or target site selection within these complementing complexes. This allowed the demonstration that the IN variant contributing the active catalytic domain was also responsible for recognition of the integration target DNA. We were further able to establish that a single monomer is responsible for both functions by use of assay mixtures containing three different IN genotypes. These data specify the ligands bound at the catalytically relevant IN monomer and allow more-specific modeling of the mechanism of inhibitors that also bind this surface of IN.

Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase

Swanwick, Richard S.; Maglia, Giovanni; Tey, Lai-hock; Allemann, Rudolf K.
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
Português
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The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0±0.2 at 5 °C to 2.2±0.2 at 40 °C and an inverse ratio of the pre-exponential factors of 0.108±0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue, Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors...