Página 1 dos resultados de 722 itens digitais encontrados em 0.733 segundos

Conformational Isomerism Can Limit Antibody Catalysis*S⃞

Debler, Erik W.; Müller, Roger; Hilvert, Donald; Wilson, Ian A.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 13/06/2008 Português
Relevância na Pesquisa
85.7%
Ligand binding to enzymes and antibodies is often accompanied by protein conformational changes. Although such structural adjustments may be conducive to enzyme catalysis, much less is known about their effect on reactions promoted by engineered catalytic antibodies. Crystallographic and pre-steady state kinetic analyses of antibody 34E4, which efficiently promotes the conversion of benzisoxazoles to salicylonitriles, show that the resting catalyst adopts two interconverting active-site conformations, only one of which is competent to bind substrate. In the predominant isomer, the indole side chain of TrpL91 occupies the binding site and blocks ligand access. Slow conformational isomerization of this residue, on the same time scale as catalytic turnover, creates a deep and narrow binding site that can accommodate substrate and promote proton transfer using GluH50 as a carboxylate base. Although 34E4 is among the best catalysts for the deprotonation of benzisoxazoles, its efficiency appears to be significantly limited by this conformational plasticity of its active site. Future efforts to improve this antibody might profitably focus on stabilizing the active conformation of the catalyst. Analogous strategies may also be relevant to other engineered proteins that are limited by an unfavorable conformational pre-equilibrium.

The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP*S⃞

Cho, Jin-Ah; Osenkowski, Pamela; Zhao, Huiren; Kim, Seaho; Toth, Marta; Cole, Kristina; Aboukameel, Amro; Saliganan, Allen; Schuger, Lucia; Bonfil, R. Daniel; Fridman, Rafael
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 20/06/2008 Português
Relevância na Pesquisa
85.67%
Membrane type 1 (MT1) matrix metalloproteinase (MMP-14) is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recombinant 44-MT1 (Gly285–Val582) in HT1080 fibrosarcoma cells results in enhanced pro-MMP-2 activation, proliferation within a three-dimensional collagen I matrix, and tumor growth and lung metastasis in mice. Stimulation of pro-MMP-2 activation and growth in collagen I was also observed in other cell systems. Expression of 44-MT1 in HT1080 cells is associated with a delay in the rate of active MT1-MMP endocytosis resulting in higher levels of active enzyme at the cell surface. Consistently, deletion of the cytosolic domain obliterates the stimulatory effects of 44-MT1 on MT1-MMP activity. In contrast...

Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins*S⃞

Ilagan, Robielyn P.; Tiso, Mauro; Konas, David W.; Hemann, Craig; Durra, Deborah; Hille, Russ; Stuehr, Dennis J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 11/07/2008 Português
Relevância na Pesquisa
85.73%
Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6–16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr...

Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor*

Valton, Julien; Mathevon, Carole; Fontecave, Marc; Nivière, Vincent; Ballou, David P.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 18/04/2008 Português
Relevância na Pesquisa
85.55%
The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O2. This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein·protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD+ from ActVB and was strongly affected by NAD+ concentration...

Force-Clamp Spectroscopy Detects Residue Co-evolution in Enzyme Catalysis*S⃞

Perez-Jimenez, Raul; Wiita, Arun P.; Rodriguez-Larrea, David; Kosuri, Pallav; Gavira, Jose A.; Sanchez-Ruiz, Jose M.; Fernandez, Julio M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 03/10/2008 Português
Relevância na Pesquisa
95.81%
Understanding how the catalytic mechanisms of enzymes are optimized through evolution remains a major challenge in molecular biology. The concept of co-evolution implicates that compensatory mutations occur to preserve the structure and function of proteins. We have combined statistical analysis of protein sequences with the sensitivity of single molecule force-clamp spectroscopy to probe how catalysis is affected by structurally distant correlated mutations in Escherichia coli thioredoxin. Our findings show that evolutionary anti-correlated mutations have an inhibitory effect on enzyme catalysis, whereas positively correlated mutations rescue the catalytic activity. We interpret these results in terms of an evolutionary tuning of both the enzyme-substrate binding process and the chemistry of the active site. Our results constitute a direct observation of distant residue co-evolution in enzyme catalysis.

A Stopped Flow Transient Kinetic Analysis of Substrate Binding and Catalysis in Escherichia coli d-3-Phosphoglycerate Dehydrogenase*S⃞

Burton, Rodney L.; Hanes, Jeremiah W.; Grant, Gregory A.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 31/10/2008 Português
Relevância na Pesquisa
75.8%
Pre-steady state, stopped flow analysis of Escherichia coli d-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, α-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD+ reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD+ reduction (3.8 s–1) is similar to the kcat of the enzyme (2–3 s–1) in that direction. However...

Nitro-oleic Acid, a Novel and Irreversible Inhibitor of Xanthine Oxidoreductase*

Kelley, Eric E.; Batthyany, Carlos I.; Hundley, Nicholas J.; Woodcock, Steven R.; Bonacci, Gustavo; Del Rio, J. Mauricio; Schopfer, Francisco J.; Lancaster, Jack R.; Freeman, Bruce A.; Tarpey, Margaret M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 26/12/2008 Português
Relevância na Pesquisa
85.59%
Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 μm, limiting both purine oxidation and formation of superoxide documentclass[10pt]{article} usepackage{amsmath} usepackage{wasysym} usepackage{amsfonts} usepackage{amssymb} usepackage{amsbsy} usepackage{mathrsfs} usepackage{pmc} usepackage[Euler]{upgreek} pagestyle{empty} oddsidemargin -1.0in egin{document} egin{equation*}({mathrm{O}}_{2}^{{ar {{cdot}}}})end{equation*}end{document}. Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, β-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly...

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 γ-Secretase Complexes*S⃞

Placanica, Lisa; Tarassishin, Leonid; Yang, Guangli; Peethumnongsin, Erica; Kim, Seong-Hun; Zheng, Hui; Sisodia, Sangram S.; Li, Yue-Ming
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 30/01/2009 Português
Relevância na Pesquisa
85.56%
γ-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Aβ42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of γ-secretase, invariably increase the Aβ42:Aβ40 ratio. However, the mechanism by which these mutations affect γ-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active γ-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Aβ42:Aβ40 ratios. These data suggest that perturbations to γ-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Aβ42:Aβ40 ratio.

Contribution of Non-catalytic Core Residues to Activity and Regulation in Protein Kinase A*

Yang, Jie; Kennedy, Eileen J.; Wu, Jian; Deal, Michael S.; Pennypacker, Juniper; Ghosh, Gourisankar; Taylor, Susan S.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 06/03/2009 Português
Relevância na Pesquisa
85.61%
Protein kinase A holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits which keep the enzyme in an inhibited state before activation by cyclic-AMP. The C-subunit folds into a conserved bi-lobal core flanked by N- and C-terminal tails. We report here characterization of a C-tail loss-of-function mutant, CF327A, and a related suppressor mutant, CF327A/K285P. Phe-327 is the only residue outside the kinase core that binds to the adenine ring of ATP, whereas Lys-285 is ∼45 Å away and lies in an AGC kinase-specific insert. The two mutations were previously identified from a yeast genetic screen, where the F327A mutation was unable to complement cell growth but mutation of K285P in the same allele rescued cell viability. We show that CF327A exhibits significant reduction in catalytic efficiency, which likely explains the observed loss-of-function phenotype. Interestingly, the additional K285P mutation does not restore kinase activity but reduces the inhibitory interaction of the double mutant with RII subunits. The additional K285P mutation, thus, helps to keep a low but uninhibited PKA activity that is sufficient for cell viability. The crystal structure of CF327A/K285P further reveals that recruitment of Phe-327 to the ATP binding pocket not only contributes to the hydrophobic pocket...

Aromatic Residues in the Catalytic Center of Chitinase A from Serratia marcescens Affect Processivity, Enzyme Activity, and Biomass Converting Efficiency*

Zakariassen, Henrik; Aam, Berit Bjugan; Horn, Svein J.; Vårum, Kjell M.; Sørlie, Morten; Eijsink, Vincent G. H.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 17/04/2009 Português
Relevância na Pesquisa
85.59%
The processive Serratia marcescens chitinases A (ChiA) and B (ChiB) are thought to degrade chitin in the opposite directions. A recent study of ChiB suggested that processivity is governed by aromatic residues in the +1 and +2 (aglycon) subsites close to the catalytic center. To further investigate the roles of aromatic residues in processivity and to gain insight into the structural basis of directionality, we have mutated Trp167, Trp275, and Phe396 in the -3, +1, and +2 subsites of ChiA, respectively, and characterized the hydrolytic activities of the mutants toward β-chitin and the soluble chitin-derivative chitosan. Although the W275A and F396A mutants showed only modest reductions in processivity, it was almost abolished by the W167A mutation. Thus, although aglycon subsites seem to steer processivity in ChiB, a glycon (-3) subsite seems to be adapted to do so in ChiA, in line with the notion that the two enzymes have different directionalities. Remarkably, whereas all three single mutants and the W167A/W275A double mutant showed reduced efficiency toward chitin, they showed up to 20-fold higher activities toward chitosan. These results show that the processive mechanism is essential for an efficient conversion of crystalline substrates but comes at a large cost in terms of intrinsic enzyme speed. This needs to be taken into account when devising enzymatic strategies for biomass turnover.

A Function for the RING Finger Domain in the Allosteric Control of MDM2 Conformation and Activity*

Wawrzynow, Bartosz; Pettersson, Susanne; Zylicz, Alicja; Bramham, Janice; Worrall, Erin; Hupp, Ted R.; Ball, Kathryn L.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 24/04/2009 Português
Relevância na Pesquisa
85.61%
The MDM2 oncoprotein plays multiple regulatory roles in the control of p53-dependent gene expression. A picture of MDM2 is emerging where structurally discrete but interdependent functional domains are linked through changes in conformation. The domain structure includes: (i) a hydrophobic pocket at the N terminus of MDM2 that is involved in both its transrepressor and E3-ubiqutin ligase functions, (ii) a central acid domain that recognizes a ubiquitination signal in the core DNA binding domain of p53, and (iii) a C-terminal C2H2C4 RING finger domain that is required for E2 enzyme-binding and ATP-dependent molecular chaperone activity. Here we show that the binding affinity of MDM2s hydrophobic pocket can be regulated through the RING finger domain and that increases in pocket affinity are reflected by a gain in MDM2 transrepressor activity. Thus, mutations within the RING domain that affect zinc coordination, but not one that inhibits ATP binding, produce MDM2 proteins that have a higher affinity for the BOX-I transactivation domain of p53 and a reduced I0.5 for p53 transrepression. An allosteric model for regulation of the hydrophobic pocket is supported by differences in protein conformation and pocket accessibility between wild-type and the RING domain mutant MDM2 proteins. Additionally the data demonstrate that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed toward its hydrophobic pocket.

H2S Biogenesis by Human Cystathionine γ-Lyase Leads to the Novel Sulfur Metabolites Lanthionine and Homolanthionine and Is Responsive to the Grade of Hyperhomocysteinemia*S⃞

Chiku, Taurai; Padovani, Dominique; Zhu, Weidong; Singh, Sangita; Vitvitsky, Victor; Banerjee, Ruma
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 24/04/2009 Português
Relevância na Pesquisa
85.55%
Although there is a growing recognition of the significance of hydrogen sulfide (H2S) as a biological signaling molecule involved in vascular and nervous system functions, its biogenesis and regulation are poorly understood. It is widely assumed that desulfhydration of cysteine is the major source of H2S in mammals and is catalyzed by the transsulfuration pathway enzymes, cystathionine β-synthase and cystathionine γ-lyase (CSE). In this study, we demonstrate that the profligacy of human CSE results in a variety of reactions that generate H2S from cysteine and homocysteine. The γ-replacement reaction, which condenses two molecules of homocysteine, yields H2S and a novel biomarker, homolanthionine, which has been reported in urine of homocystinuric patients, whereas a β-replacement reaction, which condenses two molecules of cysteine, generates lanthionine. Kinetic simulations at physiologically relevant concentrations of cysteine and homocysteine, reveal that the α,β-elimination of cysteine accounts for ∼70% of H2S generation. However, the relative importance of homocysteine-derived H2S increases progressively with the grade of hyperhomocysteinemia, and under conditions of severely elevated homocysteine (200 μm)...

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO*S⃞

Saito, Yohtaro; Ashida, Hiroki; Sakiyama, Tomoko; de Marsac, Nicole Tandeau; Danchin, Antoine; Sekowska, Agnieszka; Yokota, Akiho
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 08/05/2009 Português
Relevância na Pesquisa
85.64%
The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO2 fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg2+ for catalysis and, like RuBisCO, was stimulated by CO2. Four residues that are essential for the enolization reaction of RuBisCO, Lys175, Lys201, Asp203, and Glu204, were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys123...

Escherichia coli DNA Adenine Methyltransferase: THE STRUCTURAL BASIS OF PROCESSIVE CATALYSIS AND INDIRECT READ-OUT

Coffin, Stephanie R.; Reich, Norbert O.
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
95.79%
We have investigated the structural basis of processive GATC methylation by the Escherichia coli DNA adenine methyltransferase, which is critical in chromosome replication and mismatch repair. We determined the contribution of the orthologically conserved phosphate interactions involving residues Arg95, Asn126, Asn132, Arg116, and Lys139, which directly contact the DNA outside the cognate recognition site (GATC) to processive catalysis, and that of residue Arg137, which is not conserved and contacts the DNA backbone within the GATC sequence. Alanine substitutions at the conserved positions have large impacts on processivity yet do not impact kcat/KmDNA or DNA affinity (KDDNA). However, these mutants cause large preferences for GATC sites varying in flanking sequences when considering the pre-steady state efficiency constant kchem/KDDNA. These changes occur mainly at the level of the methylation rate constant, which results in the observed decreases in processive catalysis. Thus, processivity and catalytic efficiency (kcat/KmDNA) are uncoupled in these mutants. These results reveal that the binding energy involved in DNA recognition contributes to the assembly of the active site rather than tight binding. Furthermore, the conserved residues (Arg95...

Tetramerization and Cooperativity in Plasmodium falciparum Glutathione S-Transferase Are Mediated by Atypic Loop 113–119

Liebau, Eva; Dawood, Kutayba F.; Fabrini, Raffaele; Fischer-Riepe, Lena; Perbandt, Markus; Stella, Lorenzo; Pedersen, Jens Z.; Bocedi, Alessio; Petrarca, Patrizia; Federici, Giorgio; Ricci, Giorgio
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
85.61%
Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modulated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113–119 in the tetramerization process and in the cooperative phenomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoenzymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST...

Involvement of Ionizable Groups in Catalysis of Human Liver Glycolate Oxidase*

Pennati, Andrea; Gadda, Giovanni
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
85.72%
Glycolate oxidase is a flavin-dependent, peroxisomal enzyme that oxidizes α-hydroxy acids to the corresponding α-keto acids, with reduction of oxygen to H2O2. In plants, the enzyme participates in photorespiration. In humans, it is a potential drug target for treatment of primary hyperoxaluria, a genetic disorder where overproduction of oxalate results in the formation of kidney stones. In this study, steady-state and pre-steady-state kinetic approaches have been used to determine how pH affects the kinetic steps of the catalytic mechanism of human glycolate oxidase. The enzyme showed a Ping-Pong Bi-Bi kinetic mechanism between pH 6.0 and 10.0. Both the overall turnover of the enzyme (kcat) and the rate constant for anaerobic substrate reduction of the flavin were pH-independent at pH values above 7.0 and decreased slightly at lower pH, suggesting the involvement of an unprotonated group acting as a base in the chemical step of glycolate oxidation. The second-order rate constant for capture of glycolate (kcat/Kglycolate) and the Kd(app) for the formation of the enzyme-substrate complex suggested the presence of a protonated group with apparent pKa of 8.5 participating in substrate binding. The kcat/Koxygen values were an order of magnitude faster when a group with pKa of 6.8 was unprotonated. These results are discussed in the context of the available three-dimensional structure of GOX.

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis*

Brown, Nicholas G.; Shanker, Sreejesh; Prasad, B. V. Venkataram; Palzkill, Timothy
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
85.79%
TEM-1 β-lactamase is the most common plasmid-encoded β-lactamase in Gram-negative bacteria and is a model class A enzyme. The active site of class A β-lactamases share several conserved residues including Ser70, Glu166, and Asn170 that coordinate a hydrolytic water involved in deacylation. Unlike Ser70 and Glu166, the functional significance of residue Asn170 is not well understood even though it forms hydrogen bonds with both Glu166 and the hydrolytic water. The goal of this study was to examine the importance of Asn170 for catalysis and substrate specificity of β-lactam antibiotic hydrolysis. The codon for position 170 was randomized to create a library containing all 20 possible amino acids. The random library was introduced into Escherichia coli, and functional clones were selected on agar plates containing ampicillin. DNA sequencing of the functional clones revealed that only asparagine (wild type) and glycine at this position are consistent with wild-type function. The determination of kinetic parameters for several substrates revealed that the N170G mutant is very efficient at hydrolyzing substrates that contain a primary amine in the antibiotic R-group that would be close to the Asn170 side chain in the acyl-intermediate. In addition...

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles*

Kuhn, Misty L.; Falaschetti, Christine A.; Ballicora, Miguel A.
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
85.82%
ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (Vmax/Km) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference)...

Enzymatic Activity and Substrate Specificity of Mitogen-activated Protein Kinase p38α in Different Phosphorylation States*S⃞

Zhang, Yuan-Yuan; Mei, Zi-Qing; Wu, Jia-Wei; Wang, Zhi-Xin
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 26/09/2008 Português
Relevância na Pesquisa
85.7%
The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38α, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38α phosphorylated at both Thr-180 and Tyr-182 was 10–20-fold more active than p38α phosphorylated at Thr-180 only, whereas p38α phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Cα are all highly specific for the dephosphorylation of p38α...

The Significance of EXDD and RXKD Motif Conservation in Rel Proteins*

Sajish, Mathew; Kalayil, Sissy; Verma, Sunil Kumar; Nandicoori, Vinay Kumar; Prakash, Balaji
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 03/04/2009 Português
Relevância na Pesquisa
85.55%
Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3′-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg2+ due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5′Pα-O-Pβ bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins...