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 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 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.

The Kinetics of Reactions Catalyzed by Alkaline Phosphatase: The Effects of Added Nucleophiles

1972 ◽  
Vol 50 (12) ◽  
pp. 1360-1368 ◽  
Author(s):  
Irwin Hinberg ◽  
Keith J. Laidler
Keyword(s):  
Alkaline Phosphatase ◽  
Preceding Paper ◽  
The Other ◽  
Intestinal Alkaline Phosphatase ◽  
Michaelis Constant ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Kinetics Of Formation ◽  
Kinetics Of

An experimental study was made of the hydrolyses of phenyl phosphate and p-nitrophenyl phosphate catalyzed by chicken intestinal alkaline phosphatase. The work was done at pH 8.0 and 10.0, 25.0 °C, and an ionic strength of 0.1 M, and particular attention was paid to the kinetics of formation of the products in the presence of Tris and ethanolamine. It was found that the rates of formation of phenol or p-nitrophenol (P1) and of the phosphorylated nucleophile (P3) were dependent on the concentration of added nucleophile; on the other hand the rate of formation of phosphate (P2) and the Michaelis constant were independent of nucleophile concentration. This result cannot be reconciled with any of the mechanisms discussed in the preceding paper with the exception of mechanism VI, which is an elaboration of one proposed by Trentham and Gutfreund; mechanism VI is[Formula: see text]where W is water and N the alternative nucleophile. ES and E*S are two conformers of the enzyme–substrate complex, and E*S′ and ES′ two forms of the phosphorylated enzyme; only the latter can react with water and only the former with nucleophile.

scholarly journals Kinetics of inactivation of bovine pancreatic ribonuclease A by bromopyruvic acid

1996 ◽  
Vol 320 (1) ◽  
pp. 187-192 ◽  
Author(s):  
Ming-Hua WANG ◽  
Zhi-Xin WANG ◽  
Kang-Yuan ZHAO
Keyword(s):  
Competitive Inhibition ◽  
Ribonuclease A ◽  
Inactivation Kinetics ◽  
Ionization State ◽  
Substrate Complex ◽  
Irreversible Inhibition ◽  
Enzyme Product ◽  
Enzyme Substrate ◽  
Pancreatic Ribonuclease A ◽  
Kinetics Of

The kinetic theory of substrate reaction during the modification of enzyme activity [Duggleby (1986) J. Theor. Biol. 123, 67–80; Wang and Tsou (1990) J. Theor. Biol. 142, 531–549] has been applied to a study of the inactivation kinetics of ribonuclease A by bromopyruvic acid. The results show that irreversible inhibition belongs to a non-competitive complexing type inhibition. On the basis of the kinetic equation of substrate reaction in the presence of the inhibitor, all microscopic kinetic constants for the free enzyme, the enzyme–substrate complex and the enzyme–product complex have been determined. The non-competitive inhibition type indicates that neither the substrate nor the product affects the binding of bromopyruvic acid to the enzyme and that the ionization state of His-119 may be the same in both the enzyme–substrate and the enzyme–product complexes.

scholarly journals Kinetics of an enzyme reaction in which both the enzyme-substrate complex and the product are unstable or only the product is unstable

1994 ◽  
Vol 303 (2) ◽  
pp. 435-440 ◽  
Author(s):  
C Garrido-del Solo ◽  
F García-Cánovas ◽  
B H Havsteen ◽  
E Valero ◽  
R Varón
Keyword(s):  
Computer Simulation ◽  
Data Analysis ◽  
Kinetic Data ◽  
Enzyme Reaction ◽  
Enzyme Concentration ◽  
Substrate Complex ◽  
Use Of Data ◽  
Enzyme Substrate ◽  
Experimental Errors ◽  
Kinetics Of

A kinetic analysis of the Michaelis-Menten mechanism has been made for the case in which both the enzyme-substrate complex and the product are unstable or only the product is unstable, either spontaneously or as the result of the addition of a reagent. This analysis allows the derivation of equations which under conditions of limiting enzyme concentration relate the concentration of all of the species to the time. A kinetic data analysis is suggested, which leads to the evaluation of the kinetic parameters involved in the reaction. The analysis is based on the equation which describes the formation of products with time and one's experimental progress curves. We demonstrate the method numerically by computer simulation of the reaction with added experimental errors and experimentally by the use of data from the kinetic study of the action of tyrosinase on dopamine.

scholarly journals Partial purification and kinetics of oestriol 16α-glucuronyltransferase from the cytosol fraction of human liver

1970 ◽  
Vol 118 (4) ◽  
pp. 625-634 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Heinz Breuer
Keyword(s):  
Human Liver ◽  
Glucuronic Acid ◽  
Specific Radioactivity ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Hydroxyl Group ◽  
Partial Purification ◽  
Cytosol Fraction ◽  
Enzyme Substrate ◽  
Kinetics Of

An enzyme that conjugates the 16α-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000gav. for 120min. The oestriol 16α-glucuronyltransferase was purified 100-fold by 0–30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16α-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37°C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis–Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100μm respectively. Cu2+, Zn2+ and Hg2+ inhibited, whereas Mg2+, Mn2+ and Fe2+ stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17β, oestradiol-17α and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16α-monoglucuronide, a product of the reaction, did not inhibit the 16α-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16α-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5α-Pregnane-3α,20α-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16α-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.

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.

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