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 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.

scholarly journals Purification and reaction mechanism of the primary inhibitor of plasmin from human plasma

1978 ◽  
Vol 175 (2) ◽  
pp. 635-641 ◽  
Author(s):  
Ulla Christensen ◽  
Inge Clemmensen
Keyword(s):  
Dissociation Constant ◽  
Human Plasma ◽  
Active Site ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Comparison Of The Results ◽  
Sepharose 4B

The primary inhibitor of plasmin in human plasma was purified by a four-step procedure involving fractional (NH4)2SO4 precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on both a plasminogen–CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor–plasminogen complex was determined to be approx. 3μm at pH7.8. The reactions of the inhibitor with human plasmin and with bovine trypsin were studied. Comparison of the results obtained confirms the hypothesis previously presented, namely that the reaction of the inhibitor with plasmin involves at least two steps, the initial rapid formation of an enzyme–inhibitor complex followed by a slow irreversible transition to another complex. The results also indicate that the reaction of the inhibitor with trypsin involves just a single, irreversible step, so that this reaction seems to be less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (approx. 26μm) for 6-aminohexanoic acid is obtained for both its effect on the reaction of the inhibitor with trypsin and for competitive inhibition of trypsin. The inhibitory effect of 6-aminohexanoic acid thus seems to be due to its blocking of the active site of trypsin. In contrast with this, the inhibitory effects of l-lysine and 6-aminohexanoic acid on the inhibitor–plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a second site(s) on plasmin is discussed.

scholarly journals The iron(III)-adriamycin complex inhibits cytochrome c oxidase before its inactivation

1988 ◽  
Vol 250 (3) ◽  
pp. 827-834 ◽  
Author(s):  
B B Hasinoff ◽  
J P Davey
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Kinetic Studies ◽  
Slow Inactivation ◽  
Radical Species ◽  
Inhibitor Complex ◽  
Antitumour Drug ◽  
Strong Contrast

Cytochrome c oxidase was found to be competitively inhibited by a complex formed between Fe3+ and the cardiotoxic antitumour drug adriamycin (doxorubicin) with an inhibition constant, Ki, of 12 microM. This competitive inhibition precedes the slower Fe3+-adriamycin induced inactivation of cytochrome c oxidase. In strong contrast with this result, free adriamycin was not observed to either inhibit or inactivate cytochrome c oxidase (Ki greater than 3 mM). Since, typically, polycations are known to inhibit cytochrome c oxidase, the competitive inhibition displayed by the Fe3+-adriamycin complex may also result from its polycationic character. Cytochrome c oxidase was also inhibited by pentan-1-ol (Ki 13 mM), and kinetic studies carried out in the presence of both inhibitors demonstrated that the Fe3+-adriamycin complex and pentan-1-ol are mutually exclusive inhibitors of cytochrome c oxidase. The inhibitor pentan-1-ol was also effective in preventing the slow inactivation of cytochrome c oxidase induced by Fe3+-adriamycin, presumably by blocking its binding to the enzyme. It is postulated that the slow inactivation of cytochrome c oxidase occurs when reactive radical species are produced while the Fe3+-adriamycin is complexed to cytochrome c oxidase in an enzyme-inhibitor complex. The Fe3+-adriamycin-induced inactivation of cytochrome c oxidase may be, in part, responsible for the cardiotoxicity of adriamycin.

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.

Production by Thrombin of a Proteolytically Modified form of Antithrombin and Release of the Same form from the Antithrombin-Thrombin Complex

1979 ◽  
Author(s):  
W. Fish ◽  
I. Björk
Keyword(s):  
Protease Inhibitor ◽  
Active Site ◽  
Enzyme Inhibitor ◽  
Serine Protease Inhibitor ◽  
Prolonged Treatment ◽  
Stable Complex ◽  
Single Chain ◽  
Inhibitor Complex ◽  
Active Site Region

In addition to the in vitro formation of an inactive, stable complex (ATT) between thrombin (T) and the plasma serine protease inhibitor, antithrombin (AT), a sizeable proportion of AT is converted by T to a form which has lost the ability to inhibit T and which exhibits a reduced affinity for heparin. Under non-denaturing conditions, the isolated modified AT (ATm) could not be differentiated from native AT by hydrodynamic, electrophoretic or immunological analyses. Under reducing/denaturing conditions, ATm yielded two polypeptides, one of about 50000 and one of about 5000 daltons. Preliminary studies indicate that these are the result of a proteolytic cleavage by T of an arg-ser bond in the C-terminal end of the single-chain AT molecule. Dissociation of ATT by prolonged treatment in hot SDS or by treatment with hydoxylamine or ammonia in SDS produced only T and ATm. Under nondenaturing conditions, dissociation by the two latter agents released ATm and up to 40% of the potential T activity from ATT. However, under no conditions tested could intact AT be shown to be produced from dissociation of ATT. These results suggest that scission by T of a specific arg-ser bond in the active site region of AT, or, alternatively, conversion of this bond to a tetrahedral intermediate state, occurs during the formation of the inactive enzyme-inhibitor complex.

scholarly journals A kinetic study of the interaction between mitochondrial F1 adenosine triphosphatase and adenylyl imidodiphosphate and guanylyl imidodiphosphate

1983 ◽  
Vol 210 (3) ◽  
pp. 727-735 ◽  
Author(s):  
F J F Belda ◽  
F G Carmona ◽  
F G Cánovas ◽  
J C Gómez-Fernández ◽  
J A Lozano
Keyword(s):  
Rate Constant ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Reaction Scheme ◽  
F1 Atpase ◽  
Inhibitor Complex ◽  
Change Mechanism ◽  
Mechanism Of Hydrolysis ◽  
Rate Kinetics ◽  
Inhibited Enzyme

1. The presence of 5′-adenylyl imidodiphosphate, a non-hydrolysable analogue of ATP, in the solution used to assay the soluble bovine heart mitochondrial F1-ATPase produced slow competitive inhibition. If the enzyme was preincubated with the inhibitor before the substrate, MgATP, was added, a partial re-activation was obtained. 2. The slow inhibitory process showed first-order rate kinetics, and therefore it seems likely that a conformational change of the enzyme occurs following a faster binding process. A reaction scheme is suggested. At pH 7.8 the rate constant for the inhibition reaction was calculated to be 6.7 × 10(-2)s-1 and that for the re-activation 3.8 × 10(-3)s-1, with Keq. 17.6, indicating that the inhibited enzyme-inhibitor complex will be favoured over the non-inhibited enzyme-inhibitor complex. 3. The presence of 5′-guanylyl imidodiphosphate in the solution used to assay F1-ATPase produced rapid competitive inhibition, which was then slowly reversed until a steady state was reached. This might be explained by a rapid but reversible shift of the inhibition pathway induced by this non-hydrolysable analogue of ATP. A complex rate constant for the displacement of the inhibitor by the substrate of 7.6 × 10(-3)s-1 was calculated. 4. The results are discussed in the light of other recent observations about binding of 5′-adenylyl imidodiphosphate to F1-ATPase and with reference to the binding-site-change mechanism of hydrolysis of ATP by F1-ATPase.

scholarly journals A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

2012 ◽  
Vol 448 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Hugh P. Morgan ◽  
Martin J. Walsh ◽  
Elizabeth A. Blackburn ◽  
Martin A. Wear ◽  
Matthew B. Boxer ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Active Site ◽  
High Throughput Screening ◽  
Active Sites ◽  
Covalent Bond ◽  
Cell Types ◽  
Binding Pocket ◽  
Lysine Residue ◽  
Dependent Manner ◽  
Inhibitor Molecule

PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.

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).

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