Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier

Tian, Wei; Tsou, Chen‐Lu

doi:10.1021/bi00534a031

Cited by 354 publications

(293 citation statements)

References 10 publications

(5 reference statements)

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“…The inactivation rates (k obs ) in the presence of substrate and for different inhibitor concentrations (eight to nine inhibitor concentrations) were determined in duplicate for cathepsins O2, S and L according to Tian and Tsou [25] and as previously described…”

Section: Enzyme Assaysmentioning

confidence: 99%

Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L

Brὂmme

Klaus

Okamoto

et al. 1996

Biochemical Journal

136

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Peptidyl vinyl sulphones are a novel class of extremely potent and specific cysteine protease inhibitors. They are highly active against the therapeutically important cathepsins O2, S and L. The highest k inact \K i values exceed 10( M −" :s −" for cathepsin S and 10& M −" :s −" for cathepsins O2 and L. To study the primary specificity site of the novel human cathepsin O2 and the effectiveness of this novel class of inhibitors, a series of peptidyl vinyl sulphones with variations in the P # residue was synthesized. Leucine in the P # position was proven to be the most effective residue for cathepsin O2 and also for cathepsins S and L.

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Section: Enzyme Assaysmentioning

confidence: 99%

Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L

Brὂmme

Klaus

Okamoto

et al. 1996

Biochemical Journal

136

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“…(Kr is the dissociation equilibrium constant between chymotrypsin and II, and ki is the first order rate constant for acylation of the enzyme in the enzyme-inhibitor complex [4].) This constant is 20-50-fold greater than the values obtained with conventional power- ful inhibitors of chymotrypsin.…”

Section: Inhibition Of Chymotrypsin With Propynyl Estersmentioning

confidence: 94%

“…The absorbance change was instantaneous and in order to estimate the rate constant of the reaction between thepropynyl ester and chymotrypsin we used the method of competitive covalent inhibition of the enzyme in the presence of a substrate [4].…”

Section: Inhibition Of Enzymesmentioning

confidence: 99%

The interaction of alkynyl carboxylates with serine enzymes a potent new class of serine enzyme inhibitors

et al. 1989

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The recently reported alkynyl esters, propynyl benzoate and propynyl pmethoxybenzoate, were found to interact with a variety of serine enzymes. a-Chymotrypsin was inhibited very rapidly by an equivalent amount of the esters. Trypsin, elastase and pronase were also inhibited by the esters. On the other hand, liver esterase started to hydrolyze the alkynyl esters rapidly, but the enzyme became inhibited during the course of reaction. The inhibited enzymes exhibited slow reactivation which could be. considerably enhanced by hydroxylamine.

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“…The exponential decrease in substrate hydrolysis rate was monitored continuously for up to 5 min to an end point rate (Ͻ2% of the initial rate) with Ͻ5% consumption of substrate. Progress curves were fit by nonlinear regression to an exponential plus linear term (33,34) to obtain k obs . k obs was corrected for substrate competition by multiplying by the factor, 1 ϩ [S] 0 /K m , where [S] 0 is the substrate concentration.…”

Section: Methodsmentioning

confidence: 99%

Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme

Olson

Swanson²,

Patston

et al. 1997

Journal of Biological Chemistry

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Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3,4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thøgersen, I. B., Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859 -14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and ␤-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, ␣ 1 -proteinase inhibitor (␣ 1 PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a ratelimiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the ␣ 1 PI reactive center P1-P1 bond. Quantitation of kinetic constants for DCI and ␣ 1 PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by ␣ 1 PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.

show abstract

Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier

Cited by 354 publications

References 10 publications

Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L

Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L

The interaction of alkynyl carboxylates with serine enzymes a potent new class of serine enzyme inhibitors

Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme

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