scholarly journals GlmU (N-acetylglucosamine-1-phosphate uridyltransferase) bound to three magnesium ions and ATP at the active site

2014 ◽  
Vol 70 (6) ◽  
pp. 703-708 ◽  
Author(s):  
Neha Vithani ◽  
Vaibhav Bais ◽  
Balaji Prakash
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Magnesium Ions ◽  
Inhibitory Mechanism ◽  
Enzymatic Assays ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Family ◽  
Key Factor ◽  
Inhibitory Molecule

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme exclusive to prokaryotes, belongs to the family of sugar nucleotidyltransferases (SNTs). The enzyme binds GlcNAc-1-P and UTP, and catalyzes a uridyltransfer reaction to synthesize UDP-GlcNAc, an important precursor for cell-wall biosynthesis. As many SNTs are known to utilize a broad range of substrates, substrate specificity in GlmU was probed using biochemical and structural studies. The enzymatic assays reported here demonstrate that GlmU is specific for its natural substrates UTP and GlcNAc-1-P. The crystal structure of GlmU bound to ATP and GlcNAc-1-P provides molecular details for the inability of the enzyme to utilize ATP for the nucleotidyltransfer reaction. ATP binding results in an inactive pre-catalytic enzyme–substrate complex, where it adopts an unusual conformation such that the reaction cannot be catalyzed; here, ATP is shown to be bound together with three Mg2+ions. Overall, this structure represents the binding of an inhibitory molecule at the active site and can potentially be used to develop new inhibitors of the enzyme. Further, similar to DNA/RNA polymerases, GlmU was recently recognized to utilize two metal ions, MgA2+and MgB2+, to catalyze the uridyltransfer reaction. Interestingly, displacement of MgB2+from its usual catalytically competent position, as noted in the crystal structure of RNA polymerase in an inactive state, was considered to be a key factor inhibiting the reaction. Surprisingly, in the current structure of GlmU MgB2+is similarly displaced; this raises the possibility that an analogous inhibitory mechanism may be operative in GlmU.

scholarly journals A novel β-xylosidase structure fromGeobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

2014 ◽  
Vol 70 (5) ◽  
pp. 1366-1374 ◽  
Author(s):  
Giannina Espina ◽  
Kirstin Eley ◽  
Guillaume Pompidor ◽  
Thomas R. Schneider ◽  
Susan J. Crennell ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Glycoside Hydrolase ◽  
Thermophilic Bacterium ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Family ◽  
Hydrolase Family

Geobacillus thermoglucosidasiusis a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding aG. thermoglucosidasiusβ-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed inEscherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme–substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of theG. thermoglucosidasiusβ-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

Human cholinesterases

2020 ◽  
pp. 63-120
Author(s):  
Sergey Varfolomeev ◽  
Bella Grigorenko ◽  
Sofya Lushchekina ◽  
Alexander Nemuchin
Keyword(s):  
Active Site ◽  
The Body ◽  
Polymorphic Modification ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Mechanism Of Hydrolysis ◽  
The Central Nervous System ◽  
The Stability ◽  
Hydrolysis Of ◽  
Enzyme Reactivity

The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.

Destabilization is as important as binding

1993 ◽  
Vol 345 (1674) ◽  
pp. 3-10 ◽  
Keyword(s):  
Binding Energy ◽  
Gibbs Energy ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Large Fraction ◽  
Geometric Distortion ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Non Covalent Interactions ◽  
Covalent Interactions

Enzymes make use of non-covalent interactions with their substrates to bring about a large fraction of their catalytic activity. These interactions must destabilize, or increase the Gibbs energy, of the substrate in the active site in order that the transition state can be reached easily. This destabilization may be brought about by utilization of the intrinsic binding energy between the active site and the bound substrate by desolvation of charged groups, geometric distortion, electrostatic interactions and, especially, loss of entropy in the enzyme-substrate complex. These mechanisms are described by interaction energies and require utilization of the intrinsic binding energy that is realized from non-covalent interactions between the enzyme and substrate. Receptors and coupled vectorial processes, such as muscle contraction and active transport, utilize binding energy similarly to avoid large peaks and valleys along the Gibbs energy profile of the reaction under physiological conditions.

A Simple Model for the Enzyme-Substrate-Complex of the Quercetinase Reaction. Crystal Structure of Flavonolatocobalt(III)(salen)

1992 ◽  
Vol 47 (8) ◽  
pp. 1185-1188 ◽  
Author(s):  
Wolfgang Hiller ◽  
Akira Nishinaga ◽  
Anton Rieker
Keyword(s):  
Crystal Structure ◽  
Simple Model ◽  
Triclinic Space Group ◽  
Space Group ◽  
Substrate Complex ◽  
Enzyme Substrate

Flavonolatocobalt(III)(salen), a model enzyme-substrate-complex for the quercetinase reaction, was synthesized and its crystal structure determined. C31H23CoN2O5 · 2 CH2Cl2, M = 732.35, triclinic, space group P 1̅ , a = 12.463(2), b = 13.270(2), c = 10.041(2) Å, α = 97.89(1)°, β= 108.08(1)°, γ= 83.56(1)°, U = 1559.4 Å3, Z = 2.

Essential Arginine Residues in Lactate Dehydrogenase from Germinating Soybean

1994 ◽  
Vol 59 (2) ◽  
pp. 467-472 ◽  
Author(s):  
Jana Barthová ◽  
Irena Hulová ◽  
Miroslava Birčáková
Keyword(s):  
Enzyme Activity ◽  
Glycine Max ◽  
Lactate Dehydrogenase ◽  
Chemical Modification ◽  
Active Site ◽  
Enzyme Modification ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Arginine Residues ◽  
Blue Sepharose

The lactate dehydrogenase was isolated from soybean (Glycine max. L.) by a procedure that employed biospecific chromatography on a column of Blue-Sepharose CL-6B. The participation of the guanidine group of arginine residues in the mechanism of enzyme action was determined through kinetic and chemical modification studies. The dependence of enzyme activity on pH was followed in the alkaline region (pH 8.6 - 12.8). The pK values found were 12.4 for the enzyme substrate complex and 11.1 for the free enzyme. The enzyme was inactivated by phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and p-hydroxyphenylglyoxal reagents used in modification experiments. Kinetic analysis of the modification indicated that one arginine residue is modified when inactivation occurs. No effect was observed on the rate of inactivation upon addition of coenzyme. The extent of enzyme modification by p-hydroxyphenylglyoxal was determined. It appears there are at least two arginine residues in the active site of the enzyme.

KINETICS AND MECHANISMS OF REACTIONS CATALYZED BY PANCREATIC RIBONUCLEASE

1966 ◽  
Vol 44 (22) ◽  
pp. 2597-2610 ◽  
Author(s):  
Eileen N. Ramsden ◽  
Keith J. Laidler
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Histidine Residue ◽  
Aspartic Acid Residue ◽  
Pyrimidine Base ◽  
Substrate Complex ◽  
Imidazole Group ◽  
Enzyme Substrate ◽  
Subsequent Hydrolysis ◽  
Mechanisms Of Reactions

A kinetic study has been made of the ribonuclease-catalyzed hydrolyses of three cyclic nucleotides, cytidine-2′,3′-phosphate, uridine-2′,3′-phosphate, and N6,O5′-diacetyl cytidine-2′,3′-phosphate. Rates were measured at pH values ranging from 6 to 8.5. The variation of the kinetic parameters with pH showed that the free enzyme possesses two active groups, having pK values of 5.4 and 7.25. When the enzyme–substrate complex is formed, the pK values of the groups are increased to 6.6 and 8.4. The pK values identify these groups as imidazole groups and show that two histidine residues are present at the active site. Since both increase in pK on complex formation, it is concluded that the acid imidazole group binds the substrate, but that the basic imidazole group cannot be concerned in substrate binding and must function only in the hydrolytic step. The results indicate that the pyrimidine base is concerned in the hydrolytic step and not solely in binding, as had been postulated. It is concluded from all of the evidence that four specific sites are present at the active center of the enzyme; three are involved in binding and one in catalysis. It is proposed that the active site of ribonuclease is composed of: the histidine residue in position 12, which catalyzes the hydrolytic step; the histidine residue in position 119, which binds the 2′-ribose oxygen atom in the substrate; the lysine residue in position 41, which binds the phosphate group or anion; and the aspartic acid residue in position 121, which binds the nitrogen atom at N1 in the pyrimidine base. A mechanism for enzyme–substrate complex formation and subsequent hydrolysis is proposed.

scholarly journals KPC-2 β-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate

2020 ◽  
pp. jbc.RA120.015050
Author(s):  
Shrenik C Mehta ◽  
Ian M Furey ◽  
Orville A Pemberton ◽  
David M Boragine ◽  
Yu Chen ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Common ◽  
Covalent Intermediate ◽  
Hydrolysis Of ◽  
Carbapenem Antibiotic ◽  
Enzyme Intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases due to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared to the common TEM-1 β-lactamase. Further, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31

2014 ◽  
Vol 70 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Maryna Lahoda ◽  
Jeroen R. Mesters ◽  
Alena Stsiapanava ◽  
Radka Chaloupkova ◽  
Michal Kuty ◽  
...  
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Chloride Ions ◽  
Saturation Mutagenesis ◽  
Hydrolytic Cleavage ◽  
Wild Type ◽  
Haloalkane Dehalogenase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Resolution Structure

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme–substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme–substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

scholarly journals Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site

2014 ◽  
Vol 70 (9) ◽  
pp. 2401-2412 ◽  
Author(s):  
Vincent Cura ◽  
Nathalie Troffer-Charlier ◽  
Jean-Marie Wurtz ◽  
Luc Bonnefond ◽  
Jean Cavarelli
Keyword(s):  
Crystal Structure ◽  
Zinc Finger ◽  
Active Site ◽  
Rna Splicing ◽  
Structural Features ◽  
Arginine Methylation ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Damage Repair ◽  
The Family

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 fromMus musculusrefined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

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