scholarly journals Activation mechanism and modification kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate

1998 ◽  
Vol 335 (1) ◽  
pp. 181-189 ◽  
Author(s):  
Jia-Wei WU ◽  
Zhi-Xin WANG
Keyword(s):  
Binding Energy ◽  
Dihydrofolate Reductase ◽  
Tertiary Structure ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Activation Mechanism ◽  
Binary And Ternary Complexes ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Kinetics Of

Substrate effects on the activation kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the presence of pCMB, all modification kinetic constants for the free enzyme and enzyme–substrate binary and ternary complexes have been determined. The results of the present study indicate that the modification of Chinese hamster dihydrofolate reductase by pCMB shows single-phase kinetics, and that changes in the enzyme activity and tertiary structure proceed simultaneously during the modification process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against modification by pCMB. In the presence of a saturating concentration of NADPH, the value of kcat for 7,8-dihydrofolate in the enzyme-catalysed reaction increased four-fold on modification of Cys-6, accompanied by a two-fold increase in Km for the modified enzyme. The utilization of the binding energy of a group to increase kcat rather than reduce Km implies that the full binding energy of the group is not realized in the formation of the enzyme–substrate complex, but is used to stabilize the enzyme–transition-state complex.

scholarly journals Inactivation kinetics of dihydrofolate reductase from Chinese hamster during urea denaturation

1997 ◽  
Vol 324 (2) ◽  
pp. 395-401 ◽  
Author(s):  
Jia-Wei WU ◽  
Zhi-Xin WANG ◽  
Jun-Mei ZHOU
Keyword(s):  
Enzyme Activity ◽  
Dihydrofolate Reductase ◽  
Single Phase ◽  
Tertiary Structure ◽  
Chinese Hamster ◽  
Free Enzyme ◽  
Inactivation Kinetics ◽  
Binary And Ternary Complexes ◽  
Enzyme Substrate ◽  
Kinetics Of

The kinetic theory of substrate reaction during modification of enzyme activity has been applied to the study of inactivation kinetics of Chinese hamster dihydrofolate reductase by urea [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381–436]. On the basis of the kinetic equation of substrate reaction in the presence of urea, all microscopic kinetic constants for the free enzyme and enzyme–substrate binary and ternary complexes have been determined. The results of the present study indicate that the denaturation of dihydrofolate reductase by urea follows single-phase kinetics, and changes in enzyme activity and tertiary structure proceed simultaneously in the unfolding process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation, and enzyme–substrate complexes lose their activity less rapidly than the free enzyme.

scholarly journals Fragment-derived modulators of an industrial β-glucosidase

2020 ◽  
Vol 477 (22) ◽  
pp. 4383-4395
Author(s):  
Eleni Makraki ◽  
John F. Darby ◽  
Marta G. Carneiro ◽  
James D. Firth ◽  
Alex Heyam ◽  
...  
Keyword(s):  
Binding Site ◽  
Glycoside Hydrolase ◽  
Mixed Model ◽  
Specific Binding ◽  
Fold Increase ◽  
Kinetic Assay ◽  
Nmr Spectrum ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Maximum Activation

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme–substrate complex.

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.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

scholarly journals The effect of pH on the kinetics of arylsulphatases A and B

1983 ◽  
Vol 213 (3) ◽  
pp. 603-607 ◽  
Author(s):  
C O'Fagain ◽  
B M Butler ◽  
T J Mantle
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Substrate Binding ◽  
Acid Base ◽  
Effect Of Ph ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Kinetics Of

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.

scholarly journals Activation of dihydrofolate reductase following thiol modification involves a conformational change at the active site

1998 ◽  
Vol 335 (3) ◽  
pp. 643-646 ◽  
Author(s):  
Ying-Xin FAN ◽  
Zhen-Yu LI ◽  
Li ZHU ◽  
Jun-Mei ZHOU
Keyword(s):  
Kinetic Study ◽  
Conformational Change ◽  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Active Site ◽  
Chinese Hamster ◽  
Intrinsic Fluorescence ◽  
Activation Mechanism ◽  
Slow Phase ◽  
Ideal System

Compared with the activation of dihydrofolate reductase (DHFR) by protein denaturants and inorganic salts, activation of the enzyme by thiol modification is relatively slow. Thus it is an ideal system for kinetic study of the activation mechanism. We describe here a kinetic study of the activation of DHFRs from chicken liver and Chinese hamster ovary by p-hydroxymercuribenzoate (p-HMB). The conformational changes in the enzyme molecule that result from the modification were monitored by measuring fluorescence enhancement due to the binding of 2-p-toluidinylnaphthalene-6-sulphonate (TNS), and by monitoring changes in the intrinsic fluorescence of the enzyme. Both activation and the conformational change probed by TNS followed pseudo-first-order kinetics, and the rate constants obtained are in good agreement with each other. The change in intrinsic fluorescence is a biphasic process. The rate of the fast phase, which may reflect a change in the microenvironment of Trp-24 at the active site, coincides with the rate of activation and the conformational change probed by TNS. The rate of the slow phase, which reflects a global conformational change, is about one order of magnitude lower than that of activation. The results indicate that the activation of DHFR by p-HMB is due to modification-induced conformational changes at its active site, rather than the modification of the thiol group itself, which is almost complete within the dead-time of the experiment. This study provides kinetic evidence for the proposal that flexibility at the active site is essential for full expression of catalytic activity.

On the molecular specificity of steroid-enzyme combinations. The kinetics of β -hydroxysteroid dehydrogenase

1955 ◽  
Vol 144 (914) ◽  
pp. 116-132 ◽  
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Hydroxyl Group ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Molecular Specificity ◽  
Planar Molecules ◽  
High Concentrations ◽  
Kinetics Of ◽  
Androstane Derivatives ◽  
Marked Tendency

β -Hydroxysteroid dehydrogenase is a purified enzymic protein of bacterial origin which catalyzes oxidations of 3 β - and 17 β -hydroxysteroids to their respective ketones with diphosphopyridine nucleotide as a hydrogen acceptor. The reaction kinetics of this enzyme with a variety of steroids are not in accordance with the predictions of the theory of Michaelis & Menten (1913), since the velocity of oxidation shows a marked tendency to decline at high concentrations of substrate. The behaviour of these compounds may be fully analyzed on the assumption of the formation of an enzyme-substrate complex involving two substrate molecules. The theory for bimolecular complex formation and its implications are examined. Affinity constants have been calculated for various steroids and conclusions drawn as to the structural requirements favouring attachment to the enzyme surface. Phenolic compounds of the oestra-1:3:5(10)-triene-3-ol family are most firmly bound. Planar molecules of the androst-4-ene, androst-5-ene or 5 α -androstane series show intermediate affinity, while testane (5 β -androstane) derivatives which deviate considerably from planarity are most poorly bound to the enzyme surface. The presence of oxygenated functions at positions 3 and 17 promotes high affinity, whereas an additional 11 α - or 11 β ?-hydroxyl group opposes this effect. Conclusions have been drawn as to the manner of attachment of substrates to the enzyme surface. Certain correlations between the molecular requirements for efficient binding of steroids to the enzyme surface and their physiological activities are demonstrated.

scholarly journals Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

Biosensors ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 145
Author(s):  
Antonio Guerrieri ◽  
Rosanna Ciriello ◽  
Giuliana Bianco ◽  
Francesca De Gennaro ◽  
Silvio Frascaro
Keyword(s):  
Trichoderma Viride ◽  
Kinetic Studies ◽  
Pt Electrode ◽  
Allosteric Enzyme ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Influence ◽  
Ph Dependent ◽  
Kinetics Of ◽  
Allosteric Properties

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

A kinetic analysis of the processive enzyme Lactobacillus plantarum exoribonuclease

1977 ◽  
Vol 55 (7) ◽  
pp. 671-677 ◽  
Author(s):  
Kam-Fong Lam ◽  
David M. Logan
Keyword(s):  
Binding Energy ◽  
Lactobacillus Plantarum ◽  
Chain Length ◽  
Binding Sites ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Competitive Inhibitors ◽  
Kinetic Plots ◽  
Processive Enzyme ◽  
Almost All

Oligonucleotide chains consisting of adenosine residues and ranging from 1 to 70 residues in length have been tested as substrates or inhibitors with Lactobacillus plantarum exoribonuclease (EC 3.1.4.20). The kinetic constants V, Km, and Ki are all chain-length dependent. Ki decreases with increasing chain length to a minimum for oligonucleotides seven residues in length and then begins to increase slightly. Kinetic plots indicate that the oligonucleotides are almost all competitive inhibitors of poly A hydrolysis. However, the oligonucleotide (Ap)3A > p probably leads to mixed inhibition. The enzyme is unable to retain its processivity when it hydrolyzes short oligonucleotides such as (Ap)2A and (Ap)3A. It is proposed that L. plantarum exoribonuclease possesses seven binding sites for the polynucleotide. When the enzyme is bound to a long-chain-length substrate the complex is stabilized by a binding energy of about 8 Kcal/mol. After cleavage of the terminal nucleotide the remaining binding energy is still sufficient to maintain an enzyme–substrate complex. The shortened nucleotide chain is moved relative to the enzyme to re-form the seven-bond association by a gradient of energy of about 1.7 Kcal/mol for the change from six to seven bonds.

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