scholarly journals Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin

1976 ◽  
Vol 153 (3) ◽  
pp. 701-712 ◽  
Author(s):  
T T Wang ◽  
T Hofmann
Keyword(s):  
Binding Sites ◽  
Competitive Inhibition ◽  
Catalytic Efficiency ◽  
Acyl Transfer ◽  
High Substrate Concentration ◽  
Substrate Activation ◽  
Substrate Cleavage ◽  
Kinetics Of ◽  
Hydrolysis Of

A number of peptides were found to increase the activity of pig pepsin towards small synthetic substrates. The activators increase transpeptidation of both the acyl-transfer and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from inhibition to modest activation, but is always less than the effect on transpeptidation. The kinetics of substrate cleavage are the converse of non-competitive inhibition and show an increase in kcat. and no effect on Km values. Lineweaver-Burk plots of results obtained in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the activators. The activation is greatest at pH 4.7, less at pH 3.4, and at pH 2.0 is observable only with some of the activator peptides. The results show directly the effect of secondary binding on the catalytic efficiency of pepsin. The most effective activators are those that are most hydrophobic. The results suggest that binding in the secondary binding sites causes an increase in hydrophobicity in the catalytic site which results in increased stability of the acyl and amino intermediates, and preferential reaction with acceptors other than water. The implication that the present results strengthen the case for a role of covalent intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.

The role of N-acetylcycloserine in the acyl transfer reactions

1970 ◽  
Vol 48 (2) ◽  
pp. 218-220
Author(s):  
W. J. D. Whish ◽  
T. Viswanatha
Keyword(s):  
Acyl Transfer ◽  
Transfer Reactions ◽  
Acyl Transfer Reactions ◽  
Kinetics Of

The ability of α-N-acetylcycloserine to participate in acyl transfer reactions has been investigated. The reaction with cinnamoylimidazole yields a stable monocinnamoyl intermediate, while that with p-nitrophenyl acetate yields a derivative which undergoes facile deacylation. The kinetics of acylation of α-N-acetylcycloserine and of the deacylation of its acetyl intermediate are reported.

THE ROLE OF ALKALINE PHOSPHATASE IN INTESTINAL ABSORPTION I. THE KINETICS OF PHOSPHATASE ACTION ON VARIOUS SUBSTRATES

1953 ◽  
Vol 31 (1) ◽  
pp. 1-7
Author(s):  
Neil B. Madsen ◽  
Jules Tuba
Keyword(s):  
Alkaline Phosphatase ◽  
Average Energy ◽  
Inorganic Phosphorus ◽  
Intestinal Alkaline Phosphatase ◽  
Egg Lecithin ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of intestinal alkaline phosphatase action on sodium β-glycerophosphate, glucose 6-phosphate, and egg lecithin have been studied and compared. The Michaelis constants indicate that the enzyme shows considerably less affinity for lecithin than for the other two substrates, and the approximate ratio of activity with lecithin, glucose 6-phosphate, and sodium β-glycerophosphate is 11 : 78.5 : 100. The energies of activation for the hydrolysis of the three substrates do not differ appreciably and the average energy of activation is 14,500 calories per gram-mole. The similarity of the energies of activation together with results from inhibition studies indicate that in all probability the same enzyme is responsible for the release of inorganic phosphorus from each of the three substrates.

Modulation of Prothrombinse Assembly and Activity by Phosphotidylethanolamine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4207-4207
Author(s):  
Rinku Majumder ◽  
Xiaoe Liang ◽  
Mary Ann Quinn-Allen ◽  
Barry R. Lentz ◽  
William H. Kane
Keyword(s):  
Binding Sites ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Naturally Occurring ◽  
Early Phases

Abstract Abstract 4207 Constituents of naturally occurring phospholipid membranes regulate the activity of the prothrombinase complex. In the present study we demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PC:PE) bind factor Va with high affinity (Kd ∼10 nM) in the absence of phosphatidylserine (PS). These membranes support formation of a functional prothrombinase complex though thrombin generation at saturating factor Va concentrations is reduced approximately 60-70% compared to membranes containing 5% or more PS. The presence of PE markedly enhances the catalytic efficiency of the prothrombinase complex on membranes containing 1% PS with only modest effects on membranes containing 5% or more PS. The effect of PE on factor Va membrane binding appears to be due to direct interactions between PE and factor Va rather than to changes in membrane surface packing. Finally, we find that soluble C6PE is able to bind to factor Va (Kd ∼6.5 uM) and factor Xa (Kd ∼ 91 uM). We also show that soluble C6PE is able to stimulate formation of a partially active factor Va-factor Xa complex capable of catalyzing conversion of prothrombin to thrombin in the absence of a membrane surface. We further demonstrate that C6PE and C6PS binding sites in factor Xa are linked, as binding of one lipid enhances the binding and activity of the other. These findings provide important new insights into the role of PE in assembly of the prothrombinase complex that are relevant to understanding the activity of factor Xa on the surface of platelets particularly in the early phases of hemostasis when the concentration of PS may be limiting. Disclosures: No relevant conflicts of interest to declare.

The Kinetics of Activated Thrombin-Activatable Fibrinolysis Inhibitor with Respect to Plasminogen Binding Site Removal From Fibrin Degradation Products.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3185-3185
Author(s):  
Jonathan H. Foley ◽  
Michael E. Nesheim
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Degradation Products ◽  
Catalytic Efficiency ◽  
Plasminogen Activation ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Fibrin Degradation Products ◽  
Fibrinolysis Inhibitor ◽  
Arginine Residues ◽  
Kinetics Of

Abstract Abstract 3185 Poster Board III-122 TAFI (thrombin activatable fibrinolysis inhibitor, or carboxypeptidase U) is a plasma zymogen that can be activated by thrombin, thrombin-thrombomodulin or plasmin. When activated, TAFIa cleaves C-terminal lysine and arginine residues from plasmin modified fibrin (Fn'). Fn' as a cofactor increases the rate of plasminogen activation by 3-fold over intact fibrin and 3000-fold compared to in the absence of fibrin. Upon extensive treatment with TAFIa, the cofactor activity of TAFIa modified fibrin decreases by approximately 97%. Determining the kinetics of TAFIa will give insight into how much TAFIa is required to efficiently inhibit plasminogen activation and fibrinolysis. The kinetics of TAFIa on its primary physiological substrate were measured by exploiting the binding of plasminogen to fibrin degradation products (FDPs). Fluorescently labeled plasminogen (5IAF-Pg) was equilibrated with FDPs labeled with a quencher, QSY C5-maleimide (QSY-FDP). When 5IAF-Pg is bound to QSY-FDP a baseline fluorescence reading is obtained. When treated with TAFIa, plasminogen binding sites are removed from the QSY-FDP and the fluorescence increases. A model was used to convert the rate of fluorescence increase into the rate of Plasminogen binding site removal. The model includes two distinct binding sites on QSY-FDPs (C-terminal and internal lysines), only one of which is susceptible to removal by TAFIa (C-terminal lysine). 5IAF-Glu-Pg (fluorescent native plasminogen) binds to QSY-FDP with a Kd of 176nM and when QSY-FDP are treated with TAFIa the Kd increases to 1.06μM. It appears that 5IAF-Glu-Pg has the ability to weakly bind TAFIa-treated QSY-FDP, however, the capacity is greatly reduced. Similar binding constants were obtained for 5IAF-Lys-Pg (fluorescent plasmin-cleaved plasminogen) (Kd=92nM; Kd (+TAFIa)=1.55μM). The increase in Kd upon treatment of the QSY-FDP with TAFIa is similar to that observed with 5IAF-Glu-Pg, however, the capacity of the FDPs to bind 5IAF-Lys-Pg is relatively unchanged. The calculated rate of 5IAF-Glu-Pg binding site removal by TAFIa was determined at various QSY-FDP concentrations (0-2 μM). The data are hyperbolic in nature and when fit using the Michaelis-Menten model the kcat and Km of plasminogen binding site removal were 2.34 s-1 and 142.6nM, respectively, implying a catalytic efficiency of 16.41 μM-1s-1. The rate is sensitive to the TAFIa concentration with all TAFIa concentrations (50, 75 and 100pM) yielding similar kinetic parameters. The data described here suggest that TAFIa is very efficient in removing plasminogen binding sites. The catalytic efficiency of TAFIa toward QSY-FDP is 60-fold higher than reported for bradykinin, which was previously the best known substrate of TAFIa. This increased catalytic efficiency is due to a much lower Km (0.146 μM compared to 70.6 μM). These data are reflective of plasminogen site removal and not every C-terminal lysine or arginine cleaved by TAFIa is expected to be involved in plasminogen binding. Therefore, the catalytic efficiency of TAFIa reported here (16.41 μM-1s-1) is likely a lower limit for the true value. Disclosures No relevant conflicts of interest to declare.

Role of trinuclear Zn(II) complexes in promoting the hydrolysis of 4-nitrophenyl acetate

2004 ◽  
Vol 82 (3) ◽  
pp. 409-417 ◽  
Author(s):  
Qing-Chun Ge ◽  
Yan-He Guo ◽  
Hai Lin ◽  
Dong-Zhao Gao ◽  
Hua-Kuan Lin ◽  
...  
Keyword(s):  
Rate Constants ◽  
Volume Fraction ◽  
Bound Water ◽  
Catalytic Efficiency ◽  
Active Species ◽  
Nucleophilic Attack ◽  
Carboxyl Oxygen Atom ◽  
Ph Range ◽  
Hydrolysis Of

Potentiometric determination shows that trinuclear Zn(II) complexes of the four tripods 1,3,5-tri(2′,5′-diazahexyl)benzene (L1), 1,3,5-tri(2′,5′-diazaheptyl)benzene (L2), 1,3,5-tri(2′,5′-diazaoctyl)benzene (L3), and 1,3,5-tri(2′,5′-diazanonyl)benzene (L4) could be potential hydrolytic catalysts. CH3CN solutions containing [3Zn:L]T (0.5~2 × 10–3 mol·dm–3) with I = 0.10 mol·dm–3 of KNO3 and Good's buffer (10% volume fraction) were studied for the catalyzing hydrolysis of p-nitrophenyl acetate (NA, 0.5~2 × 10–3 mol·dm–3), at 298 K, in the 6.5–8.2 pH range. The observed rate constants, kobs, fit the equilibrium equation kobs = kcom [3Zn:L]T + kOH[OH–] + k0. The sigmoid pH~kcom profiles for NA hydrolysis suggest that either the Zn(II)-bound hydroxyl or the Zn(II)-bound water forms of the catalysts can be the active species. The observed second-order rate constants are 0.0082, 0.011, 0.0059, and 0.0019 mol–1·dm3·s–1 for the four Zn3L–H2O complexes (kA) and 0.342, 0.257, 0.382, and 0.091 mol–1·dm3·s–1 for the four Zn3L–OH- groups (kB), respectively. However, under the condition that [NA] = 0.5 × 10–3 mol·dm–3 and [3Zn:L1]T = 2~4 × 10–2 mol·dm–3 at pH 7.6, the observed rate constants, kobs, obey the equilibrium kobs = kcom[3Zn:L]T/(1/K′ + [3Zn:L]T). This indicates that the 3:1 complex (or its deprotonated hydroxide form) mediates NA hydrolysis by nucleophilic attack of the carboxyl center with the pre-formation of a coordination bond between the carboxyl oxygen atom and the Zn(II) ion. Comparison with other models was made, and the reasons for the high catalytic efficiency of the tripodal complexes were given.Key words: tripod, Zn(II), catalysis, NA hydrolysis, polynuclear.

scholarly journals Evidence for involvement of IgA1 hinge glycopeptide in the IgA1-IgA1 interaction in IgA nephropathy.

1997 ◽  
Vol 8 (6) ◽  
pp. 915-919 ◽  
Author(s):  
T Kokubo ◽  
Y Hiki ◽  
H Iwase ◽  
A Horii ◽  
A Tanaka ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Iga Nephropathy ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Hinge Region ◽  
Percent Inhibition ◽  
The Core ◽  
Proline Rich Peptide ◽  
Inhibition Curve

The study was performed to investigate the role of the IgA1 hinge region in the IgA1-IgA1 interaction, which was observed previously in IgA nephropathy. The competitive inhibition assays of the IgA1-IgA1 binding were performed using the following candidates for inhibitors: native IgA1 hinge glycopeptide (nHGP), IgA1, IgA2, and IgG. The IgA1-IgA1 binding was definitely inhibited by the nHGP and the IgA1 (maximum of percent inhibition: 66.1 and 60.5%, respectively). There was no obvious inhibition in the IgA2 and the IgG. The inhibition curves of the nHGP and the IgA1 were significantly different from that of the IgG (P < 0.01, respectively). Furthermore, to reveal the detailed binding sites in the interaction, the same inhibition assays were performed using the following substances composing the IgA1 hinge glycopeptide: galactose (Gal), N-acetyl-galactosamine (GalNAc), Gal beta 1-3GalNAc, sialic acid, tetrapeptide PTPS, and synthesized hinge proline-rich peptide PVPSTPPTPSPSTPPTPSPS (sHP). sHP, Gal beta 1-3GalNAc, Gal, and GalNAc inhibited the binding (69.3, 34.1, 14.9, 14.6%, respectively). No obvious inhibition was observed in sialic acid and tetrapeptide PTPS. The inhibition curve of sHP was significantly different from that of the PTPS (P < 0.05). Those of Gal beta 1-3GalNAc, Gal, and GalNAc were also significantly different from that of sialic acid (P < 0.05, respectively). These results suggested that the IgA1-IgA1 interaction could be mediated by the core structure including the peptide and the sugars, except for sialic acid in the hinge region, resulting in the formation of the circulating macromolecular IgA1 in IgA nephropathy.

On the Role of Magnesium in the Reaction of the Pyruvate Kinase from Salmonella typhimurium

1986 ◽  
Vol 41 (11-12) ◽  
pp. 1018-1022
Author(s):  
Concepcion Garcia-Olalla ◽  
Amando Garrido-Pertierra
Keyword(s):  
Salmonella Typhimurium ◽  
Pyruvate Kinase ◽  
Binding Sites ◽  
Physiological Significance ◽  
Hill Coefficient ◽  
Magnesium Concentration ◽  
Pyruvate Kinases ◽  
Kinetics Of ◽  
Binding Behaviour

Abstract The kinetics of the two purified forms of pyruvate kinase from Salmonella typhimurium LT-2 were studied in assays at pH 6.8 where the relationships between the initial velocities of the catalysed reactions and Mg2+ are non-hyperbolic. The analysis show that Mg2+ display positive homotropic interactions in their binding behaviour with Hill coefficient values of 2.5 and 1.2 for the form I and II, respectively. The binding sites of the cation to the pyruvate kinases seem to be independent to those for phosphoenolpyruvate and adenosine 5′-diphosphate; changes in the magnesium concentration might be of physiological significance in relation to a rapid regeneration of adenosine 5′-triphosphate by means of the pyruvate kinase reaction.

scholarly journals KINETICS OF THE CATALYZED HYDROLYSIS OF p-NITROPHENYL ACETATE

1964 ◽  
Vol 42 (11) ◽  
pp. 2404-2409 ◽  
Author(s):  
E. Sacher ◽  
K. J. Laidler
Keyword(s):  
Rate Constant ◽  
Steric Hindrance ◽  
Functional Group ◽  
Catalytic Efficiency ◽  
Bifunctional Catalysis ◽  
Simple Correlation ◽  
Hydroxide Ion ◽  
Substituted Pyridines ◽  
Kinetics Of ◽  
Hydrolysis Of

Catalytic constants have been determined for the hydrolysis at 20 °C of p-nitrophenyl acetate in 9.56% (w/w) dioxane–water mixtures; catalysts used were hydroxide ions, imidazole and 2-methylimidazole, various substituted pyridines, serine, histidine and histidylhistidine. In the case of hydroxide ion catalysis, the rate constant falls off at high catalyst concentrations, and this is attributed to the establishment of an equilibrium involving the anion of the substrate. The results with the various catalysts indicate that there is no simple correlation between catalytic efficiency and pK or nucleophilicity. In the imidazole series there is evidence of steric hindrance when a group is present in a position next to the functional group. There is an indication of bifunctional catalysis in the hydrolysis by 2-(β-hydroxyethyl)pyridine and 4-(γ-hydroxypropyl)pyridine, both of which are much more effective than pyridine.

The Role of Membrane Proteins and Phospholipids in the Interaction of Ribosomes with Endoplasmic Reticulum Membranes

1975 ◽  
Vol 53 (9) ◽  
pp. 1039-1045 ◽  
Author(s):  
Serge Jothy ◽  
Jean-Louis Bilodeau ◽  
Henry Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Molecular Weights ◽  
Low Concentrations ◽  
Phospholipid Headgroups ◽  
Hydrolysis Of

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.

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