scholarly journals Neuraminidase inhibition by chemically sulphated glycopeptides

1979 ◽  
Vol 181 (2) ◽  
pp. 377-385 ◽  
Author(s):  
N Mian ◽  
C E Anderson ◽  
P W Kent
Keyword(s):  
Enzyme Inhibition ◽  
Active Site ◽  
Enzyme Inhibitor ◽  
Helix Pomatia ◽  
Submaxillary Gland ◽  
Competitive Inhibitor ◽  
Duodenal Mucosa ◽  
Dependent Manner ◽  
Enzyme Active Site ◽  
Acetylneuraminic Acid

Chemically sulphated glycopeptides (derived from pig duodenal mucosa) inhibited Clostridium perfringens neuraminidase (EC 3.2.1.18) activity in a pH-dependent manner. Analysis of inhibition kinetics data indicated that, although the enzyme inhibition could not be categorized into any of the classical types of inhibition, it could be interpreted as a function of the size and shape of the substrates used. The enzyme activity was inhibited by 86% and 40% when tested with bovine submaxillary-gland mucin (mol. wt. 4 x 10(5)-40 x 10(5) and N-acetylneuraminyl-lactose (mol. wt. 633) as substrates respectively. Presence of sulphated glycopeptide did not affect the binding of N-acetylneuraminic acid (mol. wt. 309), a competitive inhibitor of Vibrio cholerae neuraminidase, to the enzyme active site. The enzyme inhibition was thus considered to be due to steric hindrance as a consequence of the non-specific interactions between the enzyme molecule and polyanionic sulphated glycopeptide affecting the differential accessibility of the substrate molecules to the enzyme active site. The enzyme-inhibitor interaction could be suppressed by rapid and many-fold dilution of the reaction mixture, by concurrent addition of the inactive enzyme or by partial removal of the sulphate esters from the sulphated glycopeptide molecule by the action of Helix pomatia arylsulphatase (EC 3.1.6.1).

scholarly journals Inactivation of Δ5-3-oxo steroid isomerase with active-site-directed acetylenic steroids

1981 ◽  
Vol 193 (1) ◽  
pp. 217-227 ◽  
Author(s):  
T M Penning ◽  
D F Covey ◽  
P Talalay
Keyword(s):  
Active Site ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Covalent Bond ◽  
Addition Product ◽  
Ring Structure ◽  
Dependent Manner ◽  
Compound I ◽  
Inhibitor Complex ◽  
Acetylenic Ketone

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 × 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 × 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.

scholarly journals Studies on the inhibition of ferrochelatase by N-alkylated dicarboxylic porphyrins. Steric factors involved and evidence that the inhibition is reversible

1985 ◽  
Vol 226 (2) ◽  
pp. 537-544 ◽  
Author(s):  
F De Matteis ◽  
A H Gibbs ◽  
C Harvey
Keyword(s):  
Enzyme Inhibition ◽  
Active Site ◽  
Inhibitory Activity ◽  
Alkyl Group ◽  
Structural Requirements ◽  
Prolonged Incubation ◽  
Enzyme Active Site ◽  
Steric Factors ◽  
Haem Oxygenase ◽  
Pyrrole Nitrogen

The structural requirements for the inhibition of ferrochelatase by N-alkylated porphyrins were investigated and experiments carried out to explore the mechanism of enzyme inhibition. Three dicarboxylic porphyrins, all substrates of the enzyme, are strongly inhibitory when N-alkylated; in contrast, uroporphyrin and coproporphyrin (which are not substrates) do not inhibit after N-alkylation. Free carboxylic acid functions are required for inhibition, as the methyl ester derivatives are not themselves inhibitory. Porphyrins bearing the alkyl group on the pyrrole nitrogen of rings C and D are less effective inhibitors, particularly when zinc is chelated in the centre of the tetrapyrrole or the N-alkyl group is relatively large in size. The substituents at the 2- and 4-positions of the porphyrin system may also affect the inhibitory activity, particularly for the isomers with ring C and D alkylated. The zinc chelates of several N-alkylprotoporphyrins are inhibitory towards haem oxygenase, another haem-binding enzyme, and also in this case increasing the size of the alkyl group decreased the inhibitory activity, particularly for isomers with ring C or D alkylated. The inhibition could be reversed by prolonged incubation with excess porphyrin substrate, but dealkylation of the N-alkylporphyrin during enzyme inhibition could not be demonstrated. It is concluded (a) that N-alkylated dicarboxylic porphyrins compete reversibly with the porphyrin substrate for the enzyme active site and (b) that the structural and steric factors discussed above affect the inhibitory activity by modifying the affinity of the N-alkylporphyrin inhibitor for the enzyme.

scholarly journals Studies on N-acetylneuraminic acid aldolase

1971 ◽  
Vol 125 (1) ◽  
pp. 275-284 ◽  
Author(s):  
J. E. G. Barnett ◽  
D. L. Corina ◽  
G. Rasool
Keyword(s):  
Sodium Borohydride ◽  
Active Site ◽  
Half Life ◽  
Apparent Molecular Weight ◽  
Amino Acid Derivative ◽  
Enzyme Active Site ◽  
Acetylneuraminic Acid ◽  
Bivalent Metal Ions ◽  
Mercuric Ions ◽  
Hydrolysis Of

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37°C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent Ki for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive Ki 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the ∈-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.

scholarly journals Inactivation of Recombinant Monocyte cAMP-Specific Phosphodiesterase by cAMP Analog, 8-[(4-Bromo-2,3-Dioxobutyl)thio]Adenosine 3′,5′-Cyclic Monophosphate

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1019-1026 ◽  
Author(s):  
George A. Omburo ◽  
Theodore J. Torphy ◽  
Gilbert Scott ◽  
Susanne Jacobitz ◽  
Roberta F. Colman ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Enzyme Inhibitor ◽  
Order Rate Constant ◽  
Competitive Inhibitor ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Concentration Dependent Manner ◽  
Cyclic Monophosphate ◽  
Inhibitor Complex ◽  
Camp Hydrolysis

Abstract Two cAMP analogs, 8- and 2- [(4-bromo-2,3-dioxobutyl) thio]adenosine 3′,5′-cyclic monophosphate (8- and 2-BDB-TcAMP) have been used in probing the catalytic site of recombinant monocyte cAMP-specific phosphodiesterase (PDE4a). 2-BDB-TcAMP is a reversible and competitive inhibitor (Ki = 5.5 μmol/L) of cAMP hydrolysis by PDE4a. 8-BDB-TcAMP irreversibly inactivates the enzyme in a time- and concentration-dependent manner with a second order rate constant of 0.022 mmol/L−1min−1. The rate of inactivation of PDE4a is reduced by the presence of the substrate cAMP and specific inhibitors, rolipram and denbufylline, but not by cGMP or AMP. Reduction of the enzyme-inhibitor complex with sodium [3H]borohydride shows that 1.2 mol of the affinity label/mol of enzyme was incorporated. The radiolabeled peptide is composed of 10 amino acid residues (697 to 706) located near the carboxyl end of the proposed catalytic domain. The peptide (GPGHPPLPDK) has seven nonpolar and aliphatic residues, of which four are proline, giving the peptide a highly structured conformation. This peptide is the first to be identified in the putative catalytic domain involved in substrate recognition.

scholarly journals How similar are enzyme active site geometries derived from quantum mechanical theozymes to crystal structures of enzyme-inhibitor complexes? Implications for enzyme design

2007 ◽  
Vol 16 (9) ◽  
pp. 1851-1866 ◽  
Author(s):  
Jason DeChancie ◽  
Fernando R. Clemente ◽  
Adam J.T. Smith ◽  
Hakan Gunaydin ◽  
Yi-Lei Zhao ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Enzyme Inhibitor ◽  
Quantum Mechanical ◽  
Enzyme Design ◽  
Enzyme Active Site

Active-site-directed inactivation of Schizophyllum commune cellulase by 4′, 5′-epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside

1988 ◽  
Vol 66 (8) ◽  
pp. 871-879 ◽  
Author(s):  
Anthony John Clarke
Keyword(s):  
Active Site ◽  
Schizophyllum Commune ◽  
Competitive Inhibitor ◽  
Irreversible Inhibitor ◽  
Inhibitor Molecule ◽  
Enzyme Active Site ◽  
First Order ◽  
First Order Kinetics ◽  
Pseudo First Order ◽  
Inactivation Process

4′,5′-Epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside (4) was synthesized by a Koenigs–Knorr reaction of 4-penten-1-ol and acetobromcellobiose, promoted by silver trifluoromethanesulfonate and N,N′-tetramethylurea, and tested as a potential active-site-directed irreversible inhibitor of the Schizophyllum commune cellulase. Incubation of the S. commune cellulase with 4 resulted in a time-dependent irreversible inactivation of the enzyme. The inactivation process obeyed pseudo-first-order kinetics and the hyperbolic plot of kobs as a function of inhibitor concentration provided values for Kd and k2 of 150 mM and 2.0 × 10−4 s−1, respectively, at pH 5.5 and 25 °C. The binding of a competitive inhibitor, cellobiose, to the cellulase prior to incubation with 4 protected the enzyme from rapid inactivation, suggesting that the inactivation is due to attack at the active site. The dependence of the inactivation on pH is consistent with the participation of carboxyl groups. Treatment of the affinity-labeled enzyme with [14C]methoxyamine resulted in the near stoichiometric formation of a stable radiolabelled adduct, suggesting that one inhibitor molecule binds per enzyme active site of the enzyme.

scholarly journals Active-site directed inactivation of rat ovarian 20 α-hydroxysteroid dehydrogenase

1986 ◽  
Vol 240 (3) ◽  
pp. 717-723 ◽  
Author(s):  
J W Ricigliano ◽  
T M Penning
Keyword(s):  
Active Site ◽  
Gel Filtration ◽  
Covalent Bond ◽  
Hydroxysteroid Dehydrogenase ◽  
Competitive Inhibitor ◽  
Vinyl Ketone ◽  
Allylic Alcohol ◽  
Dependent Manner ◽  
Velocity Measurements ◽  
Putative Mechanism

Rat ovarian 20 alpha-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition by catalysing the reduction of progesterone to give the progestationally inactive steroid 20 alpha-hydroxyprogesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestational maintaining agents, including the epimeric allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol and the related vinyl ketone 1-(3 beta-hydroxy-5 alpha-androstan-17 beta-yl)-2-propen-1-one. The vinyl ketone inactivates rat ovarian 20 alpha-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarose column chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivation plots gave a Ki of 2.0 microM for the inhibitor and a t1/2 for the enzyme of 20 s at saturation. These data indicate that the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis in the presence or absence of a competing nucleophile nor gel filtration reserves the inactivation, suggesting that a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20 alpha-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocity measurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketone can behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivation experiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrast the allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol are neither substrates nor inhibitors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivation observed with the vinyl ketone suggests that this compound may be useful as a progestational maintaining agent.

scholarly journals Irreversible inhibition of Δ5-3-oxosteroid isomerase by 2-substituted progesterones

1985 ◽  
Vol 226 (2) ◽  
pp. 469-476 ◽  
Author(s):  
T M Penning
Keyword(s):  
Active Site ◽  
Covalent Bond ◽  
Competitive Inhibitor ◽  
Enzyme Inactivation ◽  
Nucleophilic Attack ◽  
Dependent Manner ◽  
Compound I ◽  
Irreversible Inhibitors ◽  
Bacterial Enzyme ◽  
Oxosteroid Isomerase

2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates the isomerase after direct nucleophilic attack at the electropositive 2-position, and that tautomerization plays no role in the inactivation event. By contrast, 2-mercaptoethanol offers no protection against the inactivation mediated by 2-hydroxymethyleneprogesterone, and under the conditions of inactivation this compound appears to exist in the semi-enolized form.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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