scholarly journals Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain

2001 ◽  
Vol 276 (50) ◽  
pp. 46933-46940 ◽  
Author(s):  
Ararat J. Ablooglu ◽  
Mark Frankel ◽  
Elena Rusinova ◽  
John B. Alexander Ross ◽  
Ronald A. Kohanski
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Kinetic Properties ◽  
Activation Loop ◽  
Steady State Kinetic ◽  
Tryptic Cleavage ◽  
Loop Conformations ◽  
Steric Accessibility ◽  
Regulatory Properties

Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q ≈ D1132N ≥ D1161A > Y1162F ≈ K1085N > Q1208S ≥ wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation.

scholarly journals Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor

2015 ◽  
Vol 290 (34) ◽  
pp. 20649-20659 ◽  
Author(s):  
Kristoff T. Homan ◽  
Helen V. Waldschmidt ◽  
Alisa Glukhova ◽  
Alessandro Cannavo ◽  
Jianliang Song ◽  
...  
Keyword(s):  
Crystal Structure ◽  
G Protein ◽  
Active Site ◽  
Parent Compound ◽  
Kinase Domain ◽  
Kinetic Properties ◽  
G Protein Coupled Receptor ◽  
Structure Based Drug Design ◽  
Terminal Structure ◽  
G Protein Coupled

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

scholarly journals N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Rü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

2006 ◽  
Vol 188 (24) ◽  
pp. 8430-8440 ◽  
Author(s):  
Stephan Kolkenbrock ◽  
Katja Parschat ◽  
Bernd Beermann ◽  
Hans-Jürgen Hinz ◽  
Susanne Fetzner
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
Catalytic Triad ◽  
Kinetic Properties ◽  
Conserved Residues ◽  
Hydrolase Fold ◽  
Secondary Structure Predictions ◽  
Amide Hydrolase

ABSTRACT N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the α/β-hydrolase-fold superfamily of enzymes; inactivation of (His6-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat/Km = 208 mM−1 s−1), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His6Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His6AmqC22A/C63A markedly differed from those of His6Amq. The replacements effected some changes in Km s of the enzyme and increased k cats for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in α-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

scholarly journals dDYRK2: a novel dual-specificity tyrosine-phosphorylation-regulated kinase in Drosophila

2003 ◽  
Vol 374 (2) ◽  
pp. 381-391 ◽  
Author(s):  
Pamela A. LOCHHEAD ◽  
Gary SIBBET ◽  
Ross KINSTRIE ◽  
Tava CLEGHON ◽  
Margie RYLATT ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Kinases ◽  
Mutational Analysis ◽  
Kinase Domain ◽  
Activation Loop ◽  
Embryonic Neurogenesis ◽  
Founding Family ◽  
Dual Specificity ◽  
Eukaryotic Organisms

Dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an emerging family of protein kinases that have been identified in all eukaryotic organisms examined to date. DYRK family members are involved in regulating key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis and cellular differentiation. Two distinct subgroups exist, nuclear and cytosolic. In Drosophila, the founding family member minibrain, whose human orthologue maps to the Down syndrome critical region, belongs to the nuclear subclass and affects post-embryonic neurogenesis. In the present paper, we report the isolation of dDYRK2, a cytosolic DYRK and the putative product of the smell-impaired smi35A gene. This is the second such kinase described in Drosophila, but the first to be characterized at the molecular and biochemical level. dDYRK2 is an 81 kDa dual-specificity kinase that autophosphorylates on tyrosine and serine/threonine residues, but appears to phosphorylate exogenous substrates only on serine/threonine residues. It contains a YXY motif in the activation loop of the kinase domain in the same location as the TXY motif in mitogenactivated protein kinases. dDYRK2 is tyrosine-phosphorylated in vivo, and mutational analysis reveals that the activation loop tyrosines are phosphorylated and are essential for kinase activity. Finally, dDYRK2 is active at all stages of fly development, with elevated levels observed during embryogenesis and pupation.

α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

2001 ◽  
Vol 360 (3) ◽  
pp. 727-736 ◽  
Author(s):  
Bernd NIDETZKY ◽  
Christian EIS
Keyword(s):  
Steady State ◽  
Transition State ◽  
Schizophyllum Commune ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

scholarly journals Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Stathis D. Kotsakis ◽  
Carl-Fredrik Flach ◽  
Mohammad Razavi ◽  
D. G. Joakim Larsson
Keyword(s):  
Carbapenem Resistance ◽  
Catalytic Efficiency ◽  
Resistance Mechanisms ◽  
Broad Host Range ◽  
Evolutionary Potential ◽  
Class D ◽  
Steady State Kinetic ◽  
Natural Variant ◽  
Hydrolyzing Enzymes ◽  
Catalytic Motif

ABSTRACTWhile carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, β-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D β-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional β-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the naturalblaOXAplasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where thekcatof meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-β-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

2018 ◽  
Vol 11 (553) ◽  
pp. eaat5916 ◽  
Author(s):  
Kexin Shen ◽  
Jamie A. Moroco ◽  
Ravi K. Patel ◽  
Haibin Shi ◽  
John R. Engen ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Colony Formation ◽  
Family Members ◽  
Cellular Transformation ◽  
Soft Agar ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Mass Spectrometry Data ◽  
Activation Loop ◽  
Exchange Mass Spectrometry

Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

scholarly journals Dynamism and Cooperativity Essential for Efficient Catalysis in Aspergillus Niger Monoamine Oxidase

2019 ◽  
Author(s):  
Christian Curado-Carballada ◽  
Ferran Feixas ◽  
Sílvia Osuna
Keyword(s):  
Aspergillus Niger ◽  
Monoamine Oxidase ◽  
Active Site ◽  
Conformational Dynamics ◽  
Catalytic Efficiency ◽  
Building Blocks ◽  
Chiral Amine ◽  
Evolutionary Pathway ◽  
Accelerated Molecular Dynamics ◽  
Open States

<p><b> </b><i>Aspergillus niger </i>Monoamine Oxidase (MAO-N) is a homodimeric enzyme responsible for the oxidation of amines into the corresponding imine. Laboratory evolved variants of MAO-N in combination with a non-selective chemical reductant represents a powerful strategy for the deracemisation of chiral amine mixtures and, thus, is of interest for obtaining chiral amine building blocks. MAO-N presents a rich conformational dynamics with a flexible ß-hairpin region that can adopt closed, partially closed and open states. Despite the ß-hairpin conformational dynamics is altered along the laboratory evolutionary pathway of MAO-N, the connection between the ß-hairpin conformational dynamics and active site catalysis still remains unclear. In this work, we use accelerated molecular dynamics to elucidate the potential interplay between the ß-hairpin conformational dynamics and catalytic activity in MAO-N wild type and its evolved D5 variant. Our study reveals a delicate communication between both MAO-N subunits that impacts the active site architecture, and thus its catalytic efficiency. In both MAO-N WT and the laboratory evolved D5 variant, the ß-hairpin conformation in one of the monomers affects the productive binding of the substrate in the active site of the other subunit. However, both MAO-N WT and D5 variants show a quite different behaviour due to the distal mutations introduced experimentally with Directed Evolution. </p>

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