scholarly journals The role of magnesium ions in β-galactosidase-catalysed hydrolyses. Studies on charge and shape of the β-galactopyranosyl-binding site

1973 ◽  
Vol 133 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Gillian S. Case ◽  
Michael L. Sinnott ◽  
Jean-Pierre Tenu
Keyword(s):  
Binding Site ◽  
Equilibrium Dialysis ◽  
Competitive Inhibitor ◽  
Magnesium Ions ◽  
Pyranose Ring ◽  
Fluorescence Measurements ◽  
Tetramethylammonium Bromide ◽  
Kinetics Of ◽  
Cationic Inhibitor

1. β-d-Galactopyranosyl trimethylammonium bromide is a competitive inhibitor of β-galactosidase, Ki=1.4±0.2mm at 25°C. 2. Tetramethylammonium bromide is not an inhibitor (Ki>0.2m). 3. The kinetics of deactivation of Mg2+-saturated, and of inhibitor-and Mg2+-saturated, enzyme in 10mm-EDTA are similar. 4. The apparent Ki for the glycosylammonium salt is approx. 2.2mm in the absence of Mg2+. 5. It is therefore concluded that Mg2+ and the inhibitor bind independently, and that the Mg2+ does not act as an electrophilic catalyst. 6. Complexant fluorescence measurements indicate binding of 1 Mg2+ ion per 135000-dalton protomer. 7. This stoicheiometry is confirmed by equilibrium dialysis. 8. 1,6-Anhydrogalactopyranose is neither a substrate (kcat./Km< 3×10-2m-1·S-1) nor an inhibitor (Ki>0.2m). 9. Considerations of conformations available to the cationic inhibitor and to the anhydrogalactose indicate that the substrate is bound with the pyranose ring in a conformation not greatly different from the normal chair (C1) conformation.

scholarly journals Mitochondrial adenosine triphosphatase from human placenta--inhibition by free magnesium ions of ITP hydrolysis.

1994 ◽  
Vol 41 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Z Aleksandrowicz
Keyword(s):  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Negative Cooperativity ◽  
Magnesium Ions ◽  
Beef Liver ◽  
Bicarbonate Ions ◽  
The Hill ◽  
Kinetics Of ◽  
Free Magnesium

The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (F1) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of F1 followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial F1 seems to be the MgITP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MgITP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MgITP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MgITP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental F1 exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental F1 activity.

Role of Binding Site Loops in Controlling Nitric Oxide Release:  Structure and Kinetics of Mutant Forms of Nitrophorin 4†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (21) ◽  
pp. 6679-6690 ◽  
Author(s):  
Estelle M. Maes ◽  
Andrzej Weichsel ◽  
John F. Andersen ◽  
Donald Shepley ◽  
William R. Montfort
Keyword(s):  
Nitric Oxide ◽  
Binding Site ◽  
Nitric Oxide Release ◽  
Nitrophorin 4 ◽  
Mutant Forms ◽  
Kinetics Of

scholarly journals Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions

2021 ◽  
Author(s):  
Ali Imran ◽  
Brandon S. Moyer ◽  
Ashley J. Canning ◽  
Dan Kalina ◽  
Thomas M Duncan ◽  
...  
Keyword(s):  
Binding Site ◽  
Primary Role ◽  
Dissociation Kinetics ◽  
High Affinity ◽  
Histone 3 ◽  
Quantitative Understanding ◽  
Slow Dissociation ◽  
Kinetics Of ◽  
Association Kinetics

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of  transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

Kinetics of Action of Different Photosystem II Herbicides on Thylakoids

1990 ◽  
Vol 45 (5) ◽  
pp. 348-352 ◽  
Author(s):  
Jean-Marc Ducruet ◽  
Sophie Creuzet ◽  
Josiane Viénot
Keyword(s):  
Electron Transfer ◽  
Chlorophyll Fluorescence ◽  
Photosystem Ii ◽  
Temperature Dependent ◽  
Binding Process ◽  
Uracil Derivatives ◽  
Fluorescence Measurements ◽  
Kinetics Of

The kinctics of inhibition of photosystem II electron transfer by different diuron-like herbicides (ureas, triazines, triazinoncs, biscarbamates. uraciles) were studied, mainly by chlorophyll fluorescence measurements. Uracil derivatives and cyanazine, a particular triazinc. were the slowest acting compounds. The half-times of action were strongly temperature-dependent and were of the order of tens of seconds at 5 °C for urea or triazine inhibitors. The role of different limiting steps in the binding process is discussed.

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells

Diabetes ◽  
1997 ◽  
Vol 46 (3) ◽  
pp. 354-362 ◽  
Author(s):  
K. Matsuda ◽  
E. Araki ◽  
R. Yoshimura ◽  
K. Tsuruzoe ◽  
N. Furukawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cho Cells ◽  
Hepg2 Cells ◽  
E Box ◽  
Specific Regulation

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

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