scholarly journals Past times: The efficiency of enzyme catalysis

2003 ◽  
Vol 25 (4) ◽  
pp. 52-53
Author(s):  
Michael I. Page
Keyword(s):  
Active Site ◽  
Enzyme Catalysis ◽  
Catalytic Efficiency ◽  
Low Concentration ◽  
Rapid Turnover ◽  
Significant Rate ◽  
Diffusion Controlled ◽  
Biological Interest ◽  
Rate Of Reaction ◽  
Hydrolysis Of

Understanding enzyme catalysis on a molecular and energetic basis has fascinated scientists for more than half a century. In addition to their obvious physiological involvement, the incredible efficiency of enzymes continues to intrigue us. In the absence of enzymes, many reactions of biological interest, e.g. the hydrolysis of proteins, carbohydrates and DNA, have half-lives of hundreds to millions of years. After a substrate is bound at an enzyme's active site, its halflife is usually milliseconds. The low concentration of enzymes in cells, which is often at or below the micromolar level, means that a rapid turnover is necessary to produce a significant rate of reaction and many reactions occur at near the diffusion controlled limit. The high catalytic efficiency of enzymes has not been emulated by artificial systems and therefore many have wondered if they could even be understood by ordinary chemistry.

Formaldehyde as an Active Site Label of α-Chymotrypsin

1972 ◽  
Vol 50 (10) ◽  
pp. 1114-1121 ◽  
Author(s):  
Charles J. Martin ◽  
Narendra B. Oza ◽  
Mario A. Marini
Keyword(s):  
Active Site ◽  
Competitive Inhibition ◽  
Catalytic Efficiency ◽  
Formaldehyde Concentration ◽  
Binding Studies ◽  
Photometric Analysis ◽  
Limit Values ◽  
Catalytic Rate Constant ◽  
Catalytic Rate ◽  
Hydrolysis Of

The effect of formaldehyde concentration on the steady state parameters of the chymotrypsin-catalyzed hydrolysis of specific ester substrates has been investigated. At the limit values which are attained in about 3 M formaldehyde, the catalytic rate constant is decreased eightfold and [Formula: see text] increased ca. 2.5-foid. The reduction in activity does not conform to competitive, noncompetitive, uncompetitive, or partially competitive inhibition. The effect of increasing formaldehyde concentration is, however, in accord with an equimolar equilibrium reaction between chymotrypsin and formaldehyde with Kassoc of 5 M−1. Spectro-photometric analysis of the same reaction (J. Biol. Chem. 242, 5736 (1967)) and binding studies with 14C-elabeled formaldehyde (Biochim. Biophys. Acta, 242, 320 (1971)) have shown that both histidine residues react with formaldehyde.From a study of the imidazole group catalyzed hydrolysis of p-nitrophenyl acetate, the effect of formaldehyde on such processes is qualitatively similar to that observed for reactions catalyzed by chymotrypsin. The Kassoc values, however, are higher: 36 M−1 for imidazole and 25 M−1 for acetylhistidine.On the basis of these and other results, it is concluded that formaldehyde reacts with the active site of chymotrypsin to form a modified enzyme with decreased catalytic efficiency. The causative event is most probably due to the formation of an N-hydroxymethyl derivative of the His-57 residue.

scholarly journals Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

2019 ◽  
Vol 116 (51) ◽  
pp. 25634-25640 ◽  
Author(s):  
Medhanjali Dasgupta ◽  
Dominik Budday ◽  
Saulo H. P. de Oliveira ◽  
Peter Madzelan ◽  
Darya Marchany-Rivera ◽  
...  
Keyword(s):  
Active Site ◽  
Enzyme Catalysis ◽  
Conformational Dynamics ◽  
Catalytic Efficiency ◽  
Enzyme Mechanism ◽  
Time Resolved ◽  
X Ray ◽  
Protein Motions ◽  
X Ray Crystallography ◽  
Active Site Cysteine

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

scholarly journals Histidine protonation states are key in the LigI catalytic reaction mechanism

2021 ◽  
Author(s):  
LINA ZHAO ◽  
Dibyendu Mondal ◽  
Weifeng Li ◽  
Yuguang Mu ◽  
Philipp Kaldis
Keyword(s):  
Active Site ◽  
Enzyme Catalysis ◽  
Valence Bond ◽  
Free Energy Calculations ◽  
Active Site Residues ◽  
Protonation State ◽  
Empirical Valence Bond ◽  
Hydrolysis Of ◽  
Catalytic Reaction Mechanism

Lignin is one of the world’s most abundant organic polymers, and 2-pyrone-4,6-dicarboxylate lactonase (LigI) catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) in the degradation of lignin. The pH has profound effects on enzyme catalysis and therefore we studied this in the context of LigI. We found that changes of the pH mostly affects surface residues, while the residues at the active site are more subject to changes of the surrounding microenvironment. In accordance with this, a high pH facilitates the deprotonation of the substrate. Detailed free energy calculations by the empirical valence bond (EVB) approach revealed that the overall hydrolysis reaction is more likely when the three active site histidines (His31, His33 and His180) are protonated at the ɛ site, however, protonation at the δ site may be favored during specific steps of reaction. Our studies have uncovered the determinant role of the protonation state of the active site residues His31, His33 and His180 in the hydrolysis of PDC.

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.

Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies

2013 ◽  
Vol 454 (3) ◽  
pp. 387-399 ◽  
Author(s):  
Patrick Masson ◽  
Sofya Lushchekina ◽  
Lawrence M. Schopfer ◽  
Oksana Lockridge
Keyword(s):  
Osmotic Stress ◽  
Osmotic Pressure ◽  
Active Site ◽  
Md Simulations ◽  
Catalytic Triad ◽  
Wild Type ◽  
Irreversible Inhibitor ◽  
Enzyme Active Site ◽  
Diffusion Controlled ◽  
Peripheral Site

CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E′. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon≈108 M−1·min−1). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp70 in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium E⇌E′, indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E′. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His438, leading to the less reactive form E′.

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.

scholarly journals Extracellular α-Galactosidase from Trichoderma sp. (WF-3): Optimization of Enzyme Production and Biochemical Characterization

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Aishwarya Singh Chauhan ◽  
Arunesh Kumar ◽  
Nikhat J. Siddiqi ◽  
B. Sharma
Keyword(s):  
Enzyme Activity ◽  
Enzyme Assay ◽  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Culture Conditions ◽  
Significant Loss ◽  
Trichoderma Sp ◽  
Rate Of Reaction ◽  
Higher Temperature ◽  
Substrate Concentrations

Trichoderma spp. have been reported earlier for their excellent capacity of secreting extracellular α-galactosidase. This communication focuses on the optimization of culture conditions for optimal production of enzyme and its characterization. The evaluation of the effects of different enzyme assay parameters such as stability, pH, temperature, substrate concentrations, and incubation time on enzyme activity has been made. The most suitable buffer for enzyme assay was found to be citrate phosphate buffer (50 mM, pH 6.0) for optimal enzyme activity. This enzyme was fairly stable at higher temperature as it exhibited 72% activity at 60°C. The enzyme when incubated at room temperature up to two hours did not show any significant loss in activity. It followed Michaelis-Menten curve and showed direct relationship with varying substrate concentrations. Higher substrate concentration was not inhibitory to enzyme activity. The apparent Michaelis-Menten constant (Km), maximum rate of reaction (Vmax), Kcat, and catalytic efficiency values for this enzyme were calculated from the Lineweaver-Burk double reciprocal plot and were found to be 0.5 mM, 10 mM/s, 1.30 U mg−1, and 2.33 U mg−1 mM−1, respectively. This information would be helpful in understanding the biophysical and biochemical characteristics of extracellular α-galactosidase from other microbial sources.

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities

2014 ◽  
Vol 70 (12) ◽  
pp. 3212-3225 ◽  
Author(s):  
Tiila-Riikka Kiema ◽  
Rajesh K. Harijan ◽  
Malgorzata Strozyk ◽  
Toshiyuki Fukao ◽  
Stefan E. H. Alexson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reaction Mechanism ◽  
Active Site ◽  
Quaternary Structure ◽  
Fatty Acyl ◽  
Physiological Importance ◽  
Loop Domain ◽  
Solution Studies ◽  
Hydrolysis Of ◽  
Insight Into

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

scholarly journals Cover Feature: Distance between Metal Centres Affects Catalytic Efficiency of Dinuclear CoIII Complexes in the Hydrolysis of a Phosphate Diester (Eur. J. Org. Chem. 39/2018)

2018 ◽  
Vol 2018 (39) ◽  
pp. 5335-5335
Author(s):  
Eva Szusanna Bencze ◽  
Cristiano Zonta ◽  
Fabrizio Mancin ◽  
Leonard J. Prins ◽  
Paolo Scrimin
Keyword(s):  
Catalytic Efficiency ◽  
Phosphate Diester ◽  
Hydrolysis Of
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