Conformational Events during Ternary Enzyme−Substrate Complex Formation Are Rate Limiting in the Catalytic Cycle of the Light-Driven Enzyme Protochlorophyllide Oxidoreductase†

Biochemistry ◽  
2008 ◽  
Vol 47 (41) ◽  
pp. 10991-10998 ◽  
Author(s):  
Derren J. Heyes ◽  
Binuraj R. K. Menon ◽  
Michiyo Sakuma ◽  
Nigel S. Scrutton
Keyword(s):  
Complex Formation ◽  
Catalytic Cycle ◽  
Protochlorophyllide Oxidoreductase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting

Optical Biosensors Based on Enzyme-Substrate Complex Formation

1994 ◽  
pp. 129
Author(s):  
D.A. Moss ◽  
A. Ritter ◽  
W. Andlauer ◽  
H.J. Ache
Keyword(s):  
Complex Formation ◽  
Optical Biosensors ◽  
Substrate Complex ◽  
Enzyme Substrate

Antithrombin - Mechanism Of Action And Binding Of Heparin

1981 ◽  
Author(s):  
I Björk ◽  
U Lindahl
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Enzyme Inactivation ◽  
Substrate Complex ◽  
High Affinity ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Rate Limiting

Antithrombin inhibits a variety of serine proteases by forming equimolar, inactive complexes with the enzymes. The anti thrombin-thrombin complex, extensively studied as a model for complexes with other coagulation proteases, dissociates with a half-life of several days to free enzyme and a proteolytically modified inhibitor. It thus behaves like a kinetically stable enzyme-substrate complex. Several observations indicate that deacylation is the rate-limiting step. The active site of antithrombin, i.e. the bond slowly cleaved by the target enzyme, is the Arg-385/Ser-386 bond in the carboxy-terminal region of the protein. The formation of most anti thrombin-protease complexes is greatly accelerated by certain forms of heparin. These active molecules comprise about 1/3 of normal heparin preparations and bind with high affinity (K∼108 M-1) to the inhibitor, regardless of the size of the polysaccharide. The stoichiometry of binding is 1:1 for most heparin molecules, although some high-molecular-weight chains have two antithrombin binding sites. Evidence from spectroscopic and kinetic analyses suggests that the binding of high-affinity heparin induces a conformational change in antithrombin that probably is involved in the mechanism of the increased rate of enzyme inactivation. Oligosaccharides with high-affinity for anti thrombin have been isolated by affinity chromatography following partial deaminative cleavage of heparin with nitrous acid. The smallest such oligosaccharide obtained is an octasaccharide, in which a pentasaccharide sequence appears to comprize the actual antithrombin-binding site. This active sequence contains a unique, 3-O-sulfated glucosamine residue that does not appear to occur in other portions of the heparin molecule. In addition, two N-sulfate groups and probably at least one O-sulfate group within the pentasaccharide sequence are essential for high-affinity binding of heparin to antithrombin.

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.

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.

Total Enzyme-substrate Complex Which Includes Product-destined Enzyme-substrate Complex

2019 ◽  
pp. 1-7
Author(s):  
Ikechukwu I. Udema
Keyword(s):  
Complex Formation ◽  
Maximum Velocity ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Substrate Complex ◽  
Continuous Formation ◽  
Enzyme Substrate ◽  
The Difference ◽  
Total Enzyme

Background: There is no much interest in the determination of total enzyme-substrate complex concentration ([ES]T) which includes undissociated ES that is unaccounted for unlike the usual ES destined for transformation into free enzyme and product or substrate. The reason is speculatively as a result of the lack of awareness of such possibility via sequestration. Objectives: 1) To derive on the basis of both reverse – and standard – quasi-steady – state assumptions equations for the determination of [ES]T which is not restricted to the complex which dissociates to product/substrate and free enzyme and 2) quantitate the value of [ES]T. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to calculate relevant parameters. Results: The [EST] is < [E] ( i. e. [ET] - [ES]); [EST] decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while the velocity of amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The equations for the determination of the total enzyme-substrate complex, free enzyme without any complex formation before and after dissociation of enzyme-complex into product and/or substrate and free enzyme were derived. The difference, [ET] - [ES] is a heterogeneous mixture of undissociated ES and free enzyme without any complex formation. This is the case because [ES] which dissociates into product is only a part of the total enzyme-substrate complex. There is a continuous formation of ES during and at the expiry of the duration of assay as long as there is no total substrate depletion.

scholarly journals Association-dissociation equations, distinct from Michaelis-Menten equation for the quantification of the net flux of reactants with or without immobiliser.

2020 ◽  
Vol 13 (1) ◽  
pp. 231-243
Author(s):  
Ikechukwu Iloh Udema
Keyword(s):  
Complex Formation ◽  
Physicochemical Properties ◽  
Time Dependent ◽  
Theoretical Research ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Enzymatic Action ◽  
Immobile Phase ◽  
Net Flux ◽  
Industrial Relevance

The formation of enzyme-substrate complex, often in connection with the adsorption of the enzyme leading to either partial immobilisation in which the enzymes are adsorbed on a colloid or total immobilisation in which the enzyme is adsorbed on a rigid immobile phase is the concern of some researchers. The interest in immobilised substrate common in biological system is not very common. The objectives of this theoretical research are the rederivation of the equations of association and dissociation of reactants in the presence of adsorbents, insoluble larger macro-or supra-molecule and elucidation of why such equations are important and generalisable. The derivations produced two different equations that describe mathematically the net flux of either the substrate where the enzyme is adsorbed or the net flux of the enzyme where the substrate is adsorbed. The derivation also produced equations of translational velocities, given the probabilities that reactions occur following complex formation or that an escape of bullet molecules or dissociation reactions occur. In conclusion two different equations need separate derivation for association and dissociation of reactants. The needs for the flux of reactants have both biological and industrial relevance, respectively due to importance of time-dependent digestive processes and for the optimisation of the production of desired products of enzymatic action. The equations describing net flux seem generalisable in that information about the physicochemical properties of both crowding agent and immobilisers may not be needed for calculations.

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