scholarly journals Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase

2019 ◽  
Vol 294 (46) ◽  
pp. 17463-17470 ◽  
Author(s):  
Kyle J. Mamounis ◽  
Dante Avalos ◽  
Erik T. Yukl ◽  
Victor L. Davidson
Keyword(s):  
Active Sites ◽  
Catalytic Mechanism ◽  
Water Channel ◽  
Kinetic Mechanism ◽  
Multiple Roles ◽  
Nucleophilic Attack ◽  
Proton Relay ◽  
Initial Substrate ◽  
Stable Product ◽  
Glycine Oxidase

PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O2. Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation. In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat. This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct. These results indicate that Asp-678 is involved in the initial deprotonation of the amino group of glycine, enabling nucleophilic attack of CTQ, as well as the deprotonation of the substrate-oxidized CTQ adduct, which is coupled to CTQ reduction. The structures also suggest that Asp-678 is acting as a proton relay that directs these protons to a water channel that connects the active sites on the subunits of this homotetrameric enzyme.

scholarly journals Computational Approach Choice in Modeling Flexible Enzyme Active Sites

2019 ◽  
Author(s):  
Hisham Dokainish ◽  
James Gauld
Keyword(s):  
Conformational Changes ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Experimental Studies ◽  
Md Simulations ◽  
Acid Formation ◽  
Nucleophilic Attack ◽  
Sulfenic Acid ◽  
Proper Position

<div><div><div><p>The last step in the reductase step of the catalytic mechanism of MsrB was re-investigated using several computational approaches. Our previous QM-cluster paper showed that two possible mechanisms could occur, however the direct formation of disulfide from sulfonium cation was favored over sulfenic acid formation. In contrary, experimental studies suggest sulfenic acid formation. Therefore, first, we investigated the effect of level of theory, which confirmed previous conclusion. In addition, the effect of model choice was also investigated using ONIOM including a large QM layer around Cys440. Interestingly, deprotonating Cys440 leads to direct nucleophilic attack on Cys495 forming disulfide. Second, to eliminate the possibility that all previous results are an artifact of the used crystal structure in which the S...S distance is 3.29 Å, we ran a 5 ns MD simulation on the sulfonium cation intermediate. Surprisingly, our results show that the distance between the two sulfur is significantly increased to 4.88 Å. More importantly a water molecule is located in a proper position for nucleophilic attack. QM/MM calculations shows that sulfenic acid is formed via low barrier of 16.7 kJ mol-1. Finally, the effect of substrate binding on the two Cys's distance were investigated via running several MD simulations of possible intermediates, showing that substrate binding induces conformational changes increasing the sulfur's distance which is decreased upon substrate removal upon sulfenic acid formation. These results question the applicability of QM cluster approach in systems including flexible turns. It also emphasizes the importance of proper preparation of the starting structure.</p></div></div></div>

scholarly journals Computational Approach Choice in Modeling Flexible Enzyme Active Sites

2019 ◽  
Author(s):  
Hisham Dokainish ◽  
James Gauld
Keyword(s):  
Conformational Changes ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Experimental Studies ◽  
Md Simulations ◽  
Acid Formation ◽  
Nucleophilic Attack ◽  
Sulfenic Acid ◽  
Proper Position

<div><div><div><p>The last step in the reductase step of the catalytic mechanism of MsrB was re-investigated using several computational approaches. Our previous QM-cluster paper showed that two possible mechanisms could occur, however the direct formation of disulfide from sulfonium cation was favored over sulfenic acid formation. In contrary, experimental studies suggest sulfenic acid formation. Therefore, first, we investigated the effect of level of theory, which confirmed previous conclusion. In addition, the effect of model choice was also investigated using ONIOM including a large QM layer around Cys440. Interestingly, deprotonating Cys440 leads to direct nucleophilic attack on Cys495 forming disulfide. Second, to eliminate the possibility that all previous results are an artifact of the used crystal structure in which the S...S distance is 3.29 Å, we ran a 5 ns MD simulation on the sulfonium cation intermediate. Surprisingly, our results show that the distance between the two sulfur is significantly increased to 4.88 Å. More importantly a water molecule is located in a proper position for nucleophilic attack. QM/MM calculations shows that sulfenic acid is formed via low barrier of 16.7 kJ mol-1. Finally, the effect of substrate binding on the two Cys's distance were investigated via running several MD simulations of possible intermediates, showing that substrate binding induces conformational changes increasing the sulfur's distance which is decreased upon substrate removal upon sulfenic acid formation. These results question the applicability of QM cluster approach in systems including flexible turns. It also emphasizes the importance of proper preparation of the starting structure.</p></div></div></div>

The catalytic mechanism ofDrosophilaalcohol dehydrogenase: Evidence for a proton relay modulated by the coupled ionization of the active site Lysine/Tyrosine pair and a NAD+ribose OH switch

2003 ◽  
Vol 51 (2) ◽  
pp. 289-298 ◽  
Author(s):  
Assen Koumanov ◽  
Jordi Benach ◽  
Silvia Atrian ◽  
Roser Gonzàlez-Duarte ◽  
Andrey Karshikoff ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Proton Relay

scholarly journals A QM/MM Study of Acylphosphatase Reveals the Nucleophilic-Attack and Ensuing Carbonyl-Assisted Catalytic Mechanisms

2020 ◽  
Author(s):  
zheng zhao ◽  
Phil bourne ◽  
Hao Hu ◽  
Huanyu Chu
Keyword(s):  
Energy Barrier ◽  
Potential Energy Surfaces ◽  
Catalytic Mechanism ◽  
Interaction Networks ◽  
Nucleophilic Attack ◽  
Reaction Coordinates ◽  
Transition State Stabilization ◽  
Catalytic Mechanisms ◽  
Energy Surfaces

Acylphosphatase is one of the vital enzymes in many organs/tissues to catalyze an acylphosphate molecule into carboxylate and phosphate. Here we use a combined <i>ab initio</i> QM/MM approach to reveal the catalytic mechanism of the benzoylphosphate-bound acylphosphatase system. Using a multi-dimensional reaction-coordinates-driving scheme, we obtained a detailed catalytic process including one nucleophilic-attack and then an ensuing carbonyl-shuttle catalytic mechanism by calculating two-dimensional potential energy surfaces. We also obtained an experiment-agreeable energy barrier and validated the role of the key amino acid Asn38. Additionally, we qualified the transition state stabilization strategy based on the amino acids-contributed interaction networks revealed in the enzymatic environment. This study provided usefule insights into the underlying catalytic mechanism to contribute to disease-involved research.

scholarly journals The Role of Electrostatics in Enzymes: Do Biomolecular Force Fields Reflect Protein Electric Fields?

2020 ◽  
Author(s):  
Richard T Bradshaw ◽  
Jacek Dziedzic ◽  
Chris-Kriton Skylaris ◽  
Jonathan W. Essex
Keyword(s):  
Electric Fields ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Force Fields ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Polarizable Force Field ◽  
Polarizable Force Fields ◽  
Protein Active Sites ◽  
Dynamics Simulations

<div><div><div><p>Preorganization of large, directionally oriented, electric fields inside protein active sites has been proposed as a crucial contributor to catalytic mechanism in many enzymes, and may be efficiently investigated at the atomistic level with molecular dynamics simulations. Here we evaluate the ability of the AMOEBA polarizable force field, as well as the additive Amber ff14SB and Charmm C36m models, to describe the electric fields present inside the active site of the peptidyl-prolyl isomerase cyclophilin A. We compare the molecular mechanical electric fields to those calculated with a fully first principles quantum mechanical (QM) representation of the protein, solvent, and ions, and find that AMOEBA consistently shows far greater correlation with the QM electric fields than either of the additive force fields tested. Catalytically-relevant fields calculated with AMOEBA were typically smaller than those observed with additive potentials, but were generally consistent with an electrostatically-driven mechanism for catalysis. Our results highlight the accuracy and the potential advantages of using polarizable force fields in systems where accurate electrostatics may be crucial for providing mechanistic insights.</p></div></div></div>

scholarly journals Short hydrogen bonds in the catalytic mechanism of serine proteases

2008 ◽  
Vol 73 (4) ◽  
pp. 393-403 ◽  
Author(s):  
Vladimir Leskovac ◽  
Svetlana Trivic ◽  
Draginja Pericin ◽  
Mira Popovic ◽  
Julijan Kandrac
Keyword(s):  
Hydrogen Bond ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Serine Proteases ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Data Bank ◽  
Ground State Structure ◽  
Low Barrier Hydrogen Bonds ◽  
Short Hydrogen Bonds

The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78-1.28 ? revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 ? long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short "low-barrier" hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the "low-barrier hydrogen bond" hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 ? long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the "low-barrier hydrogen bond" hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

scholarly journals Methanol conversion on borocarbonitride catalysts: Identification and quantification of active sites

2020 ◽  
Vol 6 (26) ◽  
pp. eaba5778 ◽  
Author(s):  
Xuefei Zhang ◽  
Pengqiang Yan ◽  
Junkang Xu ◽  
Fan Li ◽  
Felix Herold ◽  
...  
Keyword(s):  
Active Sites ◽  
Density Functional ◽  
Catalytic Mechanism ◽  
Reaction Pathway ◽  
Methanol Conversion ◽  
Kinetic Measurements ◽  
Catalytic Systems ◽  
X Ray Absorption ◽  
Further Development

Borocarbonitrides (BCNs) have emerged as highly selective catalysts for the oxidative dehydrogenation (ODH) reaction. However, there is a lack of in-depth understanding of the catalytic mechanism over BCN catalysts due to the complexity of the surface oxygen functional groups. Here, BCN nanotubes with multiple active sites are synthesized for oxygen-assisted methanol conversion reaction. The catalyst shows a notable activity improvement for methanol conversion (29%) with excellent selectivity to formaldehyde (54%). Kinetic measurements indicate that carboxylic acid groups on BCN are responsible for the formation of dimethyl ether, while the redox catalysis to formaldehyde occurs on both ketonic carbonyl and boron hydroxyl (B─OH) sites. The ODH reaction pathway on the B─OH site is further revealed by in situ infrared, x-ray absorption spectra, and density functional theory. The present work provides physical-chemical insights into the functional mechanism of BCN catalysts, paving the way for further development of the underexplored nonmetallic catalytic systems.

Investigation of the Catalytic Properties of the Dysthrombin, Thrombin Quick

1979 ◽  
Author(s):  
R.A. Henriksen ◽  
W.G. Owen ◽  
M.E. Nesheim ◽  
K.G. Mann
Keyword(s):  
Active Site ◽  
Mayo Clinic ◽  
Active Sites ◽  
Fluorescence Enhancement ◽  
Catalytic Mechanism ◽  
Antithrombin Iii ◽  
Catalytic Properties ◽  
Inhibitor Binding ◽  
Equivalent Number ◽  
Aggregation Of Platelets

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and Ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of normal thrombin (T) The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. When incubated with antithrombin III, both T and TQ form inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinguished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 x 10-5M and kcat - 6.93 compared to Km = 4.0 × 10-5M and kcat = 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for tydrolysis of the active site titrant 4-methyumbel1 i feryl-p-guanidlnobenzoate.

scholarly journals Crystal structure of archaeal phosphopantothenate synthetase

2014 ◽  
Vol 70 (a1) ◽  
pp. C455-C455
Author(s):  
Akiko Kita ◽  
Asako Kishimoto ◽  
Takuya Ishibashi ◽  
Hiroya Tomita ◽  
Yuusuke Yokooji ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Rotation Axis ◽  
Structural Features ◽  
Common Pathway ◽  
Hyperthermophilic Archaeon ◽  
Pantothenate Synthetase ◽  
Almost All ◽  
Coenzyme A Biosynthesis

Bacteria/eukaryotes share a common pathway for coenzyme A biosynthesis which involves two enzymes, pantothenate synthetase and pantothenate kinase, to convert pantoate to 4'-phosphopantothenate. These two enzymes are absent in almost all archaea. Recently, it was reported that two novel enzymes, pantoate kinase (PoK) and phosphopantothenate synthetase (PPS), are responsible for this conversion in archaea[1]. In archaea, pantoate is phosphorylated by PoK to produce 4-phosphopantoate (PPo), and then condensation of PPo and β-alanine is catalyzed by PPS, generating 4'-phosphopantothenate. Here, we report the crystal structure of PPS from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complexes with ATP, and ATP and 4-phosphopantoate (PPo). PPS forms an asymmetric homodimer, in which two monomers composing a dimer, deviated from the exact 2-fold symmetry, displaying 40-130distortion. Two active sites in PPS dimer are located near the rotation axis. Due to the asymmetricity of PPS dimer molecule, two active sites in PPS dimer are not equivalent. The structural features are consistent with the mutagenesis data and the results of biochemical experiments previously reported. Based on the structures of PPS, PPS/ATP complex, and PPS/ATP/PPo complex, we discuss the catalytic mechanism by which PPS produces phosphopantoyl adenylate (PPA), which is thought to be a reaction intermediate.

scholarly journals The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

2001 ◽  
Vol 276 (15) ◽  
pp. 11698-11704 ◽  
Author(s):  
Pär L. Pettersson ◽  
Bengt Mannervik
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Ph Dependence ◽  
Enzymatic Catalysis ◽  
Catalytic Efficiency ◽  
Protonated Form ◽  
Hydroxyl Group ◽  
Isomerization Reaction

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Δ5-androstene-3,17-dione (AD) into Δ4-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect.S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr9into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pKavalue of the enzyme-bound glutathione thiol. Thus, Tyr9promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr9residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3β-hydroxysteroid dehydrogenase.

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