Página 15 dos resultados de 8928 itens digitais encontrados em 0.010 segundos

Catalysis of lysine 48-specific ubiquitin chain assembly by residues in E2 and ubiquitin

Rodrigo-Brenni, Monica C.; Foster, Scott A.; Morgan, David O.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 27/08/2010 Português
Relevância na Pesquisa
26.52%
Protein ubiquitination is catalyzed by ubiquitin-conjugating enzymes (E2s) in collaboration with ubiquitin-protein ligases (E3s). This process depends on nucleophilic attack by a substrate lysine on a thioester bond linking the C-terminus of ubiquitin to a cysteine in the E2 active site. Different E2 family members display specificity for lysines in distinct contexts. We addressed the mechanistic basis for this lysine selectivity in Ubc1, an E2 that catalyzes the ubiquitination of lysine 48 (K48) in ubiquitin, leading to the formation of K48-linked polyubiquitin chains. We identified a cluster of polar residues near the Ubc1 active site, as well as a residue in ubiquitin itself, that are required for catalysis of K48-specific ubiquitin ligation but not for general activity toward other lysines. Our results suggest that the active site of Ubc1, as well as the surface of ubiquitin, contain specificity determinants that channel specific lysines to the central residues involved directly in catalysis.

Exploring challenges in rational enzyme design by simulating the catalysis in artificial kemp eliminase

Frushicheva, Maria P.; Cao, Jie; Chu, Zhen T.; Warshel, Arieh
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
One of the fundamental challenges in biotechnology and in biochemistry is the ability to design effective enzymes. Doing so would be a convincing manifestation of a full understanding of the origin of enzyme catalysis. Despite an impressive progress, most of the advances on this front have been made by placing the reacting fragments in the proper places, rather than by optimizing the environment preorganization, which is the key factor in enzyme catalysis. Rational improvement of the preorganization would require approaches capable of evaluating reliably the actual catalytic effect. This work takes apreviously designed kemp eliminases as a benchmark for a computer aided enzyme design, using the empirical valence bond as the main screening tool. The observed absolute catalytic effect and the effect of directed evolution are reproduced and analyzed (assuming that the substrate is in the designed site). It is found that, in the case of kemp eliminases, the transition state charge distribution makes it hard to exploit the active site polarity, even with the ability to quantify the effect of different mutations. Unexpectedly, it is found that the directed evolution mutants lead to the reduction of solvation of the reactant state by water molecules rather that to the more common mode of transition state stabilization used by naturally evolved enzymes. Finally it is pointed out that our difficulties in improving Kemp eliminase are not due to overlooking exotic effect...

Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis: A CHITINASE WITHOUT CHITIN BINDING AND INSERTION DOMAINS*

Hsieh, Yin-Cheng; Wu, Yue-Jin; Chiang, Tzu-Ying; Kuo, Chueh-Yuan; Shrestha, Keshab Lal; Chao, Cheng-Fu; Huang, Yen-Chieh; Chuankhayan, Phimonphan; Wu, Wen-guey; Li, Yaw-Kuen; Chen, Chun-Jung
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)n, into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67—Thr-69 and Ile-106–Val-112) to interact with (NAG)n, generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ∼4.6 Å along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)n reveal (NAG)2, (NAG)2, and (NAG)4 in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)4; in an intermediate state, E145Q/Y227F with a boat-form NAG at the −1 subsite, −1-(NAG); after hydrolysis, E145Q with a chair form −1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between −1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of −1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of −1-(NAG). Additionally...

Cysteine 351 is an Essential Nucleophile in Catalysis by Porphyromonas gingivalis Peptidylarginine Deiminase★

Rodríguez, Sofía B.; Stitt, Barbara L.; Ash, David E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
Peptidylarginine deiminase (PAD), which catalyzes the deimination of the guanidino group from peptidylarginine residues, belongs to a superfamily of guanidino-group modifying enzymes that have been shown to produce an S-alkylthiouronium ion intermediate during catalysis. Thiol-directed reagents iodoacetamide and iodoacetate inactivate recombinant PAD, and substrate protects the enzyme from inactivation. Activity measurements together with peptide mapping by mass spectrometry of PAD modified in the absence and presence of substrate demonstrated that cysteine-351 is modified by iodoacetamide. The pKa value of the cysteine residue, 7.7 ± 0.2 as determined by iodoacetamide modification, agrees well with a critical pK value identified in pH rate studies. The role of cysteine-351 in catalysis was tested by site-directed mutagenesis in which the cysteine was replaced with serine to eliminate the proposed nucleophilic interaction. Binding studies carried out using fluorescence spectrometry established the structural integrity of the C351S PAD. However, the C351S PAD variant was catalytically inactive, exhibiting <0.01% wild-type activity. These results indicate that Cys 351 is a nucleophile that initiates the enzymatic reaction.

Catalysis: transition-state molecular recognition?

Williams, Ian H
Fonte: Beilstein-Institut Publicador: Beilstein-Institut
Tipo: Artigo de Revista Científica
Publicado em 03/11/2010 Português
Relevância na Pesquisa
26.52%
The key to understanding the fundamental processes of catalysis is the transition state (TS): indeed, catalysis is a transition-state molecular recognition event. Practical objectives, such as the design of TS analogues as potential drugs, or the design of synthetic catalysts (including catalytic antibodies), require prior knowledge of the TS structure to be mimicked. Examples, both old and new, of computational modelling studies are discussed, which illustrate this fundamental concept. It is shown that reactant binding is intrinsically inhibitory, and that attempts to design catalysts that focus simply upon attractive interactions in a binding site may fail. Free-energy changes along the reaction coordinate for SN2 methyl transfer catalysed by the enzyme catechol-O-methyl transferase are described and compared with those for a model reaction in water, as computed by hybrid quantum-mechanical/molecular-mechanical molecular dynamics simulations. The case is discussed of molecular recognition in a xylanase enzyme that stabilises its sugar substrate in a (normally unfavourable) boat conformation and in which a single-atom mutation affects the free-energy of activation dramatically.

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

Wilson, Timothy J.; Lilley, David M.J.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /02/2011 Português
Relevância na Pesquisa
26.52%
The active centers of the hairpin and VS ribozymes are both generated by the interaction of two internal loops, and both ribozymes use guanine and adenine nucleobases to accelerate cleavage and ligation reactions. The centers are topologically equivalent and the relative positioning of key elements the same. There is good evidence that the cleavage reaction of the VS ribozyme is catalyzed by the guanine (G638) acting as general base and the adenine (A756) as general acid. We now critically evaluate the experimental mechanistic evidence for the hairpin ribozyme. We conclude that all the available data are fully consistent with a major contribution to catalysis by general acid–base catalysis involving the adenine (A38) and guanine (G8). It appears that the two ribozymes are mechanistically equivalent.

Structure and Catalysis of Acylaminoacyl Peptidase: CLOSED AND OPEN SUBUNITS OF A DIMER OLIGOPEPTIDASE*

Harmat, Veronika; Domokos, Klarissza; Menyhárd, Dóra K.; Palló, Anna; Szeltner, Zoltán; Szamosi, Ilona; Beke-Somfai, Tamás; Náray-Szabó, Gábor; Polgár, László
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted...

Membrane catalysis of peptide-receptor binding

Langelaan, David N.; Rainey, Jan K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/2010 Português
Relevância na Pesquisa
26.52%
The membrane catalysis hypothesis states that a peptide ligand activates its target receptor after an initial interaction with the surrounding membrane. Upon membrane binding and interaction, the ligand is structured such that receptor binding and activation is encouraged. As evidence for this hypothesis, there are numerous studies concerning the conformation that peptides adopt in membrane mimetic environments. This mini-review analyzes the features of ligand peptides with available high-resolution membrane-induced structure and a characterized membrane-binding region. At the peptide-membrane interface, both amphipathic helices and turn structures are commonly formed in peptide ligands and both hydrophobic and electrostatic interactions can be responsible for membrane binding. Apelin is the ligand to the G-protein coupled receptor (GPCR) named APJ, with various important physiological effects, which we have recently characterized both in solution and bound to anionic micelles. The structural changes that apelin undergoes when binding to micelles provide strong evidence for membrane catalysis of apelin-APJ interactions.

Accumulation of Tetrahedral Intermediates in Cholinesterase Catalysis: A Secondary Isotope Effect Study

Tormos, Jose R.; Wiley, Kenneth L.; Wang, Yi; Fournier, Didier; Masson, Patrick; Nachon, Florian; Quinn, Daniel M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
In a previous communication, kinetic β-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate (Tormos, J. R., et al. (2005) JACS 127, 14538–14539). In this paper additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL3COSCH2CH2N+Me3; L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic β -deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: D3kcat/Km = 0.95 ± 0.03, D3kcat = 1.12 ± 0.02 and D3 β kcat = 0.97 ± 0.04. The inverse isotope effect on kcat/Km is consistent with conversion of the sp2 hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis...

Haloacetamidine-based inactivators of Protein Arginine Deiminase 4 (PAD4): Evidence that General Acid Catalysis Promotes Efficient Inactivation**

Knuckley, Bryan; Causey, Corey P.; Pellechia, Perry J.; Cook, Paul F.; Thompson, Paul R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/01/2010 Português
Relevância na Pesquisa
26.52%
Dysregulated Protein Arginine Deiminase (PAD) activity, particularly PAD4, has been suggested to play a role in the onset and progression of numerous human diseases, including Rheumatoid Arthritis (RA). Given the potential role of PAD4 in RA, we set out to develop inhibitors/inactivators that could be used to modulate PAD activity and disease progression. This effort led to the discovery of two mechanism-based inactivators, denoted F- and Cl-amidine, that inactivate PAD4 via the covalent modification of an active site cysteine that is critical for catalysis. To gain further insights into the mechanism of inactivation by these compounds, the effect of pH on the rates of inactivation were determined. These results, combined with the results of solvent isotope effect and proton inventory studies, strongly suggest that the inactivation of PAD4 by F- and Cl-amidine proceeds via a multi-step mechanism that involves the protonation and stabilization of the tetrahedral intermediate formed upon nucleophilic attack by the active site cysteine, i.e. Cys645. Stabilization of this intermediate would help to drive the halide-displacement reaction, which results in the formation of a three-membered sulfonium ring that ultimately collapses to form the inactivated enzyme. This finding - that protonation of the tetrahedral intermediate is important for enzyme inactivation - may also suggest that during catalysis...

Value of General Acid–Base Catalysis to Ribonuclease A

Thompson, James E.; Raines, Ronald T.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1994 Português
Relevância na Pesquisa
26.52%
Bovine pancreatic ribonuclease A (RNase A) is a much studied enzyme that efficiently catalyzes the cleavage of RNA. The active site of RNase A contains two histidine residues with imidazole groups positioned to act as a general base (H12) and a general acid (H119) during catalysis of RNA cleavage. Recombinant DNA techniques were used to produce mutant enzymes in which either H12 or H119 was replaced with an alanine residue. Each mutation resulted in a 104-fold decrease in the value of kcat/Km for cleaving either poly(C) or UpA. Thus, H12 and H119 each lower by 5–6 kcal/mol the free energy of the rate-limiting transition state during RNA cleavage. The value of kcat/Km for cleavage of UpOC6H4-p-NO2 was decreased by 104-fold by replacing H12 but was unaffected by replacing H119. This result provides the first direct evidence that H119 acts as a general acid during catalysis by RNase A.

Trinuclear Metal Clusters in Catalysis by Terpenoid Synthases

Aaron, Julie A.; Christianson, David. W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
Relevância na Pesquisa
26.52%
Terpenoid synthases are ubiquitous enzymes that catalyze the formation of structurally and stereochemically diverse isoprenoid natural products. Many isoprenoid coupling enzymes and terpenoid cyclases from bacteria, fungi, protists, plants, and animals share the class I terpenoid synthase fold. Despite generally low amino acid sequence identity among these examples, class I terpenoid synthases contain conserved metal binding motifs that coordinate to a trinuclear metal cluster. This cluster not only serves to bind and orient the flexible isoprenoid substrate in the precatalytic Michaelis complex, but it also triggers the departure of the diphosphate leaving group to generate a carbocation that initiates catalysis. Additional conserved hydrogen bond donors assist the metal cluster in this function. Crystal structure analysis reveals that the constellation of three metal ions required for terpenoid synthase catalysis is generally identical among all class I terpenoid synthases of known structure.

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine*

Cottrell, Joseph W.; Scott, Lincoln G.; Fedor, Martha J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pKa values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pKa for ionization of 8azaA at position 38 with the apparent pKa for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pKa values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5′-oxygen–phosphorus bond.

Trapping Conformational States Along Ligand-Binding Dynamics of Peptide Deformylase: The Impact of Induced Fit on Enzyme Catalysis

Fieulaine, Sonia; Boularot, Adrien; Artaud, Isabelle; Desmadril, Michel; Dardel, Frédéric; Meinnel, Thierry; Giglione, Carmela
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally “zipped up” by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A “movie” that reconstructs this entire process can be further extrapolated to catalysis.

Hydrogen-Bonding Catalysis and Inhibition by Simple Solvents in the Stereoselective Kinetic Epoxide-Opening Spirocyclization of Glycal Epoxides to Form Spiroketals

Wurst, Jacqueline M.; Liu, Guodong; Tan, Derek S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
Mechanistic investigations of a MeOH-induced kinetic epoxide-opening spirocyclization of glycal epoxides have revealed dramatic, specific roles for simple solvents in hydrogen-bonding catalysis of this reaction to form spiroketal products stereoselectively with inversion of configuration at the anomeric carbon. A series of electronically-tuned C1-aryl glycal epoxides was used to study the mechanism of this reaction based on differential reaction rates and inherent preferences for SN2 versus SN1 reaction manifolds. Hammett analysis of reaction kinetics with these substrates is consistent with an SN2 or SN2-like mechanism (ρ = −1.3 vs. ρ = −5.1 for corresponding SN1 reactions of these substrates). Notably, the spirocyclization reaction is second-order dependent on MeOH and the glycal ring oxygen is required for second-order MeOH catalysis. However, acetone cosolvent is a first-order inhibitor of the reaction. A transition state consistent with the experimental data is proposed in which one equivalent of MeOH activates the epoxide electrophile via a hydrogen bond while a second equivalent of MeOH chelates the sidechain nucleophile and glycal ring oxygen. A paradoxical previous observation that decreased MeOH concentration leads to increased competing intermolecular methyl glycoside formation is resolved by the finding that this side reaction is only first-order dependent on MeOH. This study highlights the unusual abilities of simple solvents to act as hydrogen-bonding catalysts and inhibitors in epoxide-opening reactions...

Required Levels of Catalysis for Emergence of Autocatalytic Sets in Models of Chemical Reaction Systems

Hordijk, Wim; Kauffman, Stuart A.; Steel, Mike
Fonte: Molecular Diversity Preservation International (MDPI) Publicador: Molecular Diversity Preservation International (MDPI)
Tipo: Artigo de Revista Científica
Publicado em 12/05/2011 Português
Relevância na Pesquisa
26.52%
The formation of a self-sustaining autocatalytic chemical network is a necessary but not sufficient condition for the origin of life. The question of whether such a network could form “by chance” within a sufficiently complex suite of molecules and reactions is one that we have investigated for a simple chemical reaction model based on polymer ligation and cleavage. In this paper, we extend this work in several further directions. In particular, we investigate in more detail the levels of catalysis required for a self-sustaining autocatalytic network to form. We study the size of chemical networks within which we might expect to find such an autocatalytic subset, and we extend the theoretical and computational analyses to models in which catalysis requires template matching.

A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis

Bhabha, Gira; Lee, Jeeyeon; Ekiert, Damian C.; Gam, Jongsik; Wilson, Ian A.; Dyson, H. Jane; Benkovic, Stephen J.; Wright, Peter E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 08/04/2011 Português
Relevância na Pesquisa
26.52%
Conformational dynamics play a key role in enzyme catalysis. While protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of E. coli dihydrofolate reductase (DHFR) that abrogates millisecond time scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. Remarkably, this dynamic knockout severely impairs hydride transfer. Thus we have found a link between conformational fluctuations on the millisecond timescale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for attempts to design novel protein catalysts.

A Transient Kinetic Analysis of PRMT1 Catalysis

Feng, You; Xie, Nan; Jin, Miyeong; Stahley, Mary R.; Stivers, James T.; Zheng, Yujun George
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
Posttranslational modifications (PTMs) are important strategies used by eukaryotic organisms to modulate their phenotypes. One of the well studied PTMs, arginine methylation, is catalyzed by protein arginine methyltransferases (PRMTs) with SAM as the methyl donor. The functions of PRMTs have been broadly studied in different biological processes and diseased states, but the molecular basis for arginine methylation is not well defined. In this study, we report the transient-state kinetic analysis of PRMT1 catalysis. The fast association and dissociation rates suggest that PRMT1 catalysis of histone H4 methylation follows a rapid equilibrium sequential kinetic mechanism. The data give direct evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, from the stopped-flow fluorescence measurements, we have identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. These results provide new insights into the mechanism of arginine methylation and the rational design of PRMT inhibitors.

Membrane Inlet for Mass Spectrometric Measurement of Catalysis by Enzymatic Decarboxylases

Moral, Mario E. G.; Tu, Chingkuang; Richards, Nigel G. J.; Silverman, David N.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the non-oxidative reaction, but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO2 and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO2 from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO2 using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O2 using MIMS also estimates the oxidase activity. Use of isotope-labeled substrate (13C2-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product 13CO2 (m/z 45), avoiding inaccuracies due to endogenous 12CO2.

Structural and Enzymatic Insights into Caspase-2 Protein Substrate Recognition and Catalysis*

Tang, Yinyan; Wells, James A.; Arkin, Michelle R.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
26.52%
Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4- and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.