scholarly journals Catalytic properties of alkaline phosphatase from pig kidney

1974 ◽  
Vol 141 (1) ◽  
pp. 283-291 ◽  
Author(s):  
Kunio Hiwada ◽  
Ernst D. Wachsmuth
Keyword(s):  
Alkaline Phosphatase ◽  
Enzymic Activity ◽  
Ph Optimum ◽  
Active Centre ◽  
Substrate Complex ◽  
Pig Kidney ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (Vmax.) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme–substrate complex is 8.7 for p-nitrophenyl phosphate and β-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme–substrate complex, 8.7, is very similar to that for the enzyme–substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (ΔH) at pH9.5 showed a transition at 24.8°C that was unaffected by Mg2+. 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn2+ ions/tetrameric enzyme for an ordered association of the monomers. Zn2+ in excess and other bivalent ions compete for a second site with Mg2+. Mg2+ enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.

scholarly journals Estimation of the dissociation constants of enzyme-substrate complexes from steady-state measurements. Interpretation of pH-independence of Km

1976 ◽  
Vol 153 (2) ◽  
pp. 455-461 ◽  
Author(s):  
A Cornish-Bowden
Keyword(s):  
Dissociation Constants ◽  
Michaelis Constant ◽  
Ph Profile ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Catalytic Constant ◽  
Almost All ◽  
Rate Limiting

If the Michaelis constant of an enzyme-catalysed reaction is independent of pH under conditions where the catalytic constant varies with pH, it is equal to the thermodynamic dissociation constant of the enzyme-substrate complex. This is true for realistic mechanisms in which binding and catalytic steps, are clearly distinguished, as well as for the simpler mechanisms that have been considered previously. It is also true for a mechanism in which a bell-shaped pH profile for the catalytic constant results from a change of rate-limiting step with pH. The relaxation time for ionization of a typical group in unbuffered solutions at 25 degrees C is of the order of 0.1 ms at the longest, and is much shorter in buffered solutions. Thus ionizations in almost all enzyme mechanisms can properly be treated as equilibria, provided that ionization is not accompanied by a slow, compulsory change in conformation.

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.

scholarly journals The carboxybiotin complex of pyruvate carboxylase. A kinetic analysis of the effects of Mg2+ ions on its stability and on its reaction with pyruvate

1984 ◽  
Vol 219 (1) ◽  
pp. 243-251 ◽  
Author(s):  
P V Attwood ◽  
J C Wallace ◽  
D B Keech
Keyword(s):  
Pyruvate Carboxylase ◽  
General Scheme ◽  
Order Rate Constant ◽  
Enzymic Activity ◽  
Slow Phase ◽  
First Order ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Pseudo First Order ◽  
Rate Limiting

The enzyme-[14C] carboxybiotin complex of sheep liver pyruvate carboxylase was isolated and the reaction between this and pyruvate was studied by using the quenched-flow rapid-reaction technique. At 0.5 degrees C the reaction was 80% complete within 180 ms. The reaction was monophasic and obeyed pseudo-first-order kinetics. Increasing concentrations of Mg2+ caused a decrease in the magnitude of the observed pseudo-first-order rate constant. Throughout the carboxylation of pyruvate, the rate-limiting step of the reaction occurred after the dissociation of carboxybiotin from the first sub-site, whereas in the slow phase of the reaction with 2-oxobutyrate this dissociation is the rate-limiting step. It is possible, from the reaction scheme proposed, that the inhibition of overall enzymic activity by high concentrations of Mg2+ could be caused by the transfer of the carboxy group from biotin to pyruvate becoming rate-limiting. The efficacy of a substrate as a signal for the movement of carboxybiotin from the first sub-site is reflected by the amount that the effective affinity of the enzyme- carboxybiotin complex for Mg2+ is lowered. In the presence of the substrates tested, the affinities of the carboxybiotin complex can be arranged in order of increasing magnitude, i.e.: (formula; see text). The kinetics of the decay of the enzyme-[14C] carboxybiotin complex at 0 degree C in the absence of substrates are similar to the reaction with pyruvate except that the carboxybiotin is also unstable in the first sub-site, to some degree. This similarity allows for the proposal of a general scheme for the decarboxylation of the enzyme- carboxybiotin complex in the presence or in the absence of substrates.

Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

1978 ◽  
Vol 39 (02) ◽  
pp. 496-503 ◽  
Author(s):  
P A D’Amore ◽  
H B Hechtman ◽  
D Shepro
Keyword(s):  
Endothelial Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Aortic Endothelial Cells ◽  
Ornithine Decarboxylase Activity ◽  
Rate Limiting Step ◽  
Bovine Aortic Endothelial Cells ◽  
Limiting Step ◽  
Rate Limiting ◽  
Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

Dynamics of hepatic and peripheral insulin effects suggest common rate-limiting step in vivo

Diabetes ◽  
1993 ◽  
Vol 42 (2) ◽  
pp. 296-306 ◽  
Author(s):  
D. C. Bradley ◽  
R. A. Poulin ◽  
R. N. Bergman
Keyword(s):  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting
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