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 Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues

2004 ◽  
Vol 378 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Krisztina PAAL ◽  
Makoto ITO ◽  
Stephen G. WITHERS
Keyword(s):  
Kinetic Studies ◽  
Acid Base ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Dependent Inactivation ◽  
Paenibacillus Sp ◽  
Continuous Assay ◽  
Reduced Activity ◽  
Enzyme Intermediate

GCase (glucosylceramidase) from Paenibacillus sp. TS12, a family 3 glycosidase, hydrolyses the β-glycosidic linkage of glucosylceramide with retention of anomeric configuration via a two-step, double-displacement mechanism. Two carboxyl residues are essential for catalysis, one functioning as a nucleophile and the other as a general acid/base catalyst. p-Nitrophenyl β-d-glucopyranoside [Km=0.27±0.02 mM and kcat/Km=(2.1±0.2)×106 M−1·s−1] and 2,4-dinitrophenyl β-d-glucopyranoside [Km=0.16±0.02 mM and kcat/Km=(2.9±0.4)×106 M−1·s−1] were used for continuous assay of the enzyme. The dependence of kcat (and kcat/Km) on pH revealed a dependence on a group of pKa≤7.8 in the enzyme–substrate complex which must be protonated for catalysis. Incubation of GCase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranoside caused time-dependent inactivation (Ki=2.4±0.7 mM and ki=0.59±0.05 min−1) due to the accumulation of a trapped glycosyl–enzyme intermediate. Electrospray ionization MS analysis of the peptic digest of this complex showed that the enzyme was covalently labelled by the reagent at Asp-223, consistent with its role as nucleophile. A mutant modified at this residue (D223G) showed substantially reduced activity compared with the wild type (>104), but this activity could be partially restored by addition of formate as an external nucleophile. Kinetic analysis of the mutant E411A indicated that Glu-411 serves as the general acid/base catalytic residue since this mutant was pH-independent and since considerable GCase activity was restored upon addition of azide to E411A, along with formation of a glycosyl azide product.

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.

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.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document