Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors

Schechter, Norman M.; Sprows, Jennifer L.; Schoenberger, Oeyvind L.; Lazarus, Gerald S.; Cooperman, Barry S.; Rubin, Harvey

doi:10.1016/s0021-9258(19)30080-8

Cited by 126 publications

(11 citation statements)

References 34 publications

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“…It is more likely that the two observed cleavages in the RSL represent competition between two sites on a2-AP for reaction with chymotrypsin (Figure 4). Such a competition has been reported during the reaction of human chymase with al-antichymotrypsin and al-PI (Schechter et al, 1989) in which cleavage could occur several residues upstream or downstream of the reactive centre. The competition was influenced by pH and ionic strength.…”

Section: Discussionmentioning

confidence: 59%

An examination of the inhibitory mechanism of serpins by analysing the interaction of trypsin and chymotrypsin with α2-antiplasmin

Enghild

Valnickova

Thøgersen

et al. 1993

Biochemical Journal

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Human alpha 2-antiplasmin (alpha 2-AP) has previously been shown to possess overlapping inhibitory sites for trypsin and chymotrypsin [Potempa, Shieh and Travis (1988) Science 241, 699-700]. Since this is currently unique among active-site-directed inhibitors of proteinases, and difficult to explain in terms of accepted inhibitory mechanisms, we re-examined the claim. Initial characterization of purified alpha 2-AP revealed an additional 12 residues preceding the published N-terminus, prompting us to revise the previous numbering. We found that trypsin caused cleavage of the Arg376-Met377 bond in the reactive-site loop of the inhibitor, whereas chymotrypsin caused cleavage at two sites in approx. equal amounts at 37 degrees C: Met374-Ser375 (site 1) and Met377-Ser378 (site 2). At 0 degrees C alpha 2-AP became a more efficient inhibitor of chymotrypsin, and the proportion of cleavage at site 1 declined, indicating that chymotrypsin prefers to react with site 2 at 0 degrees C. Inhibitors of the alpha 2-AP type are inactivated when cleaved in their reactive-site loops by proteinases that they do not inhibit, so we conclude that site 1 is treated as a substrate by chymotrypsin. Site 2 is the inhibitory site for chymotrypsin. We confirm that alpha 2-AP does indeed have overlapping reactive sites for trypsin and chymotrypsin, and since the locations of chymotrypsin-interaction sites vary with temperature, we suggest that alpha 2-AP cannot have rigid reactive-site geometry. More likely, it has a mobile reactive-site loop of the type that has been recently demonstrated for eglin C.

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Section: Discussionmentioning

confidence: 59%

An examination of the inhibitory mechanism of serpins by analysing the interaction of trypsin and chymotrypsin with α2-antiplasmin

Enghild

Valnickova

Thøgersen

et al. 1993

Biochemical Journal

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“…N-Terminal Sequence Analysis of C-Terminal Peptides Released from the Reaction of HNE with ACT and ACT Variants. The C-terminal peptide fragment was isolated and sequenced as previously described (Schechter et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Conversion of .alpha.1-Antichymotrypsin into a Human Neutrophil Elastase Inhibitor: Demonstration of Variants with Different Association Rate Constants, Stoichiometries of Inhibition, and Complex Stabilities

Rubin¹,

Plotnick²,

Wang³

et al. 1994

Biochemistry

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Despite the homology with alpha 1-protease inhibitor (alpha 1PI), wild-type antichymotrypsin (ACT) is a substrate for HNE rather than an inhibitor of the enzyme. In order to investigate the nature of the specificity between serpins and serine proteases, the reactions of human neutrophil elastase (HNE) with wild-type recombinant ACT and recombinant variants of ACT were studied. ACT variants were generated where (1) the primary interaction site, the P1 position, was replaced with the P1 residue of alpha 1PI, (2) the residues corresponding to P3-P3' were replaced with those of alpha 1PI, and (3) the residues corresponding to the canonical recognition sequence as well as flanking residues encompassing the exposed reactive loop of the inhibitor were replaced with the corresponding residues of alpha 1PI. Each variant was analyzed to determine the effect of the replacements on reactions with human neutrophil elastase and chymotrypsin with regard to (1) the second-order rate constant for enzyme-serpin complex formation, (2) the number of moles of serpin required to completely inhibit 1 mol of enzyme (the stoichiometry of inhibition, SI), and (3) the stability of the enzyme-serpin complex. Replacing Leu with Met in the P1 position (rACT-L358M) was sufficient to convert rACT into an inhibitor of HNE with an apparent second-order rate constant (k'/[I]) of 4 x 10(4) M-1 s-1 and an SI of 5. The high SI was due to a concurrent hydrolytic reaction at sites in the reactive loop.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…«280nm for Trp and Tyr are 5600 and 1420 M-1 cm-1, respectively (Creighton, 1984). The concentration of human chymase was determined using its specific activity (Schechter et al, 1989). The concentrations of trypsin and chymotrypsin were determined by active-site titration with NPGB and TCI, respectively (Chase & Shaw, 1970;Schonbaum et al, 1961).…”

Section: Methodsmentioning

confidence: 99%

Human skin tryptase: Kinetic characterization of its spontaneous inactivation

Schechter¹,

Eng²,

McCaslin³

1993

Biochemistry

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The spontaneous loss of human tryptase hydrolytic activity was investigated. Time course studies monitoring the loss in catalytic activity were biphasic and correlated with a reduction in the concentration of catalytic sites. There was an initial rapid phase leading to greater than 85% loss in activity. The remaining activity gradually decayed toward completion over a 40-h period. The initial phase could be described as a first-order process with a t1/2 of approximately 6.0 min in 0.2 M NaCl (pH 6.8, 30 degrees C). The rate constant for this phase showed little, if any, sensitivity to changes in enzyme concentration, consistent with a first-order process, and analysis of the reaction as a function of temperature was consistent with a single rate-determining step. The rate of this process, however, showed marked sensitivity to changes in NaCl concentration and pH. Increasing the NaCl concentration as well as decreasing the pH below the pI (pH 6.3) reduced the rate of activity loss, whereas increasing the pH above pH 8.0 markedly increased the rate of activity loss. The effect of NaCl concentration and pH on the rate of activity loss suggests that the rate-limiting step governing the fast phase of the reaction involves electrostatic interactions. The presence of a fast and a slow phase in the decay process may suggest heterogeneity in the sample or the rapid formation of an inactive, but reversible, intermediate. A reversible intermediate was demonstrated when "inactivated tryptase" was incubated in the presence of heparin, and an increase in tryptase catalytic activity was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

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Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors

Cited by 126 publications

References 34 publications

An examination of the inhibitory mechanism of serpins by analysing the interaction of trypsin and chymotrypsin with α2-antiplasmin

An examination of the inhibitory mechanism of serpins by analysing the interaction of trypsin and chymotrypsin with α2-antiplasmin

Conversion of .alpha.1-Antichymotrypsin into a Human Neutrophil Elastase Inhibitor: Demonstration of Variants with Different Association Rate Constants, Stoichiometries of Inhibition, and Complex Stabilities

Human skin tryptase: Kinetic characterization of its spontaneous inactivation

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