p-Guanidinobenzoic acid esters of fluorescein as active-site titrants of serine proteases

Melhado, L. Lee; Peltz, Stuart W.; Leytus, Steven P.; Mangel, Walter F.

doi:10.1021/ja00389a065

Cited by 136 publications

(44 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The acyl-intermediate complexes were prepared by incubating 100 nM proteinase with fluorescein mono-p-guanidinobenzoate at 140 nM for ϳ15 min in the case of trypsin, at 5 M for 8 min in the case of thrombin, and at 5 M for 100 min in the case of factor Xa, all at 25°C in 30 mM TAPS, 30 mM Hepes, 15 mM MES, 0.1 M NaCl, 1 mM EDTA, 0.1% PEG 8000, pH 7.4. These times were sufficient to maximally form the acyl-enzyme complex in keeping with reported rate constants (37,43). Complexes were then extensively diluted into 400 M substrates in the same mixed buffer adjusted to the desired pH at 37°C and the 405-nm absorbance was continuously monitored as for the serpin-proteinase complex dissociation experiments.…”

Section: Methodsmentioning

confidence: 99%

The pH Dependence of Serpin-Proteinase Complex Dissociation Reveals a Mechanism of Complex Stabilization Involving Inactive and Active Conformational States of the Proteinase Which Are Perturbable by Calcium

Calugaru

Swanson

Olson

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function is defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpinproteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes.Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k diss, app ) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k diss, app for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of tosylarginine methyl ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10 -100 mM Ca 2؉ ions stimulated up to 80-fold the dissociation rate constant of several serpintrypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpinproteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational changeinduced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca 2؉

show abstract

Section: Methodsmentioning

confidence: 99%

The pH Dependence of Serpin-Proteinase Complex Dissociation Reveals a Mechanism of Complex Stabilization Involving Inactive and Active Conformational States of the Proteinase Which Are Perturbable by Calcium

Calugaru

Swanson

Olson

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Fractions containing the major fluorescent peak eluting at ϳ36% CH 3 CN were neutralized by addition of 1 M NH 4 HCO 3 to 50 mM and lyophilized. The purified peptide was dissolved in water, and its concentration was determined from the fluorescein absorbance at 490 nm in 10 mM NaOH with an absorption coefficient of 89,125 M Ϫ1 cm Ϫ1 (38). Purified [5F]Hir 54 -65 eluted from the above column with a linear gradient as a single peak at 39% CH 3 CN.…”

Section: Methodsmentioning

confidence: 99%

Inactivation of Thrombin by Antithrombin Is Accompanied by Inactivation of Regulatory Exosite I

Bock¹,

Olson²,

Björk³

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

to thrombin was correlated quantitatively with partial inhibition of the rate of the thrombin-antithrombin reaction, maximally decreasing the bimolecular rate constants 1.7-and 2.1-fold, respectively. These results support a mechanism in which thrombin and the thrombin-Hir 54 -65 complex can associate with antithrombin and undergo formation of the covalent thrombin-antithrombin complex at modestly different rates, with inactivation of exosite I leading to dissociation of the peptide occurring subsequent to the ratelimiting inactivation of thrombin. This mechanism may function physiologically in localizing the activity of thrombin by allowing inactivation of thrombin that is bound in exosite I-mediated complexes with regulatory proteins, such as thrombomodulin and fibrin, without prior dissociation of these complexes. Concomitant with inactivation of thrombin, the thrombin-antithrombin complex may be irreversibly released due to exosite I inactivation.The activity of the blood clotting enzyme, thrombin, is determined by a balance between proteolytic action on physiological substrates, and irreversible inactivation by the serpin, antithrombin. The substrate specificity of thrombin is regulated by macromolecular effectors which interact with the enzyme at either of two electropositive sites, exosites I and II (1, 2). Exosite I has been defined in crystallographic studies as the site occupied by the extended COOH-terminal sequence of the specific thrombin inhibitor, hirudin (3-5). Exosite II is a distinct site which binds heparin and other ligands (1). Specific binding of COOH-terminal hirudin peptides, particularly N-acetyl-hirudin 53-64 (Hirugen 1 ; Ref. 6), to thrombin has been used to establish an important functional role for exosite I in mediating thrombin interactions. The peptide inhibits thrombin hydrolysis of the substrates, fibrinogen (7), factors V and VIII (8), and activation of the thrombin receptor (9, 10); it dissociates thrombin from regulatory interactions with fibrin I and II (7, 11) and thrombomodulin (12, 13); and it inhibits thrombin inactivation by the serpin, heparin cofactor II (14,15). By contrast to these interactions, the rate of thrombin inactivation by antithrombin is decreased only 1.9-fold by Hirugen (7), contributing to the conclusion that recognition of antithrombin by thrombin does not involve exosite I significantly (7,(15)(16)(17)(18). Binding of regulatory macromolecules to exosite I results in similarly modest effects on the rate of thrombin inactivation by antithrombin. Thrombin binding to fibrin I increases the rate 2. 8-fold (19, 20), while fibrin II reduces the rate 1.6-fold (21). The rate of inactivation of thrombin bound to thrombomodulin is unaffected or decreased ϳ2-fold for thrombomodulin lacking covalently attached chondroitin sulfate (22-25), and increased 2-8-fold for thrombomodulin containing the attached glycosaminoglycan (23)(24)(25)(26)(27).The information available indicates that the catalytic site of thrombin is accessible to antithrombin when the proteinase i...

show abstract

“…Concentrations of uPA and plasmin were determined by active-site titration with fluorescein-mono-p-guanidinobenzoate (Melhado et al, 1982;Bock et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Analogs of Human Plasminogen That Are Labeled with Fluorescence Probes at the Catalytic Site of the Zymogen

Bock

Day

Verhamme

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

p-Guanidinobenzoic acid esters of fluorescein as active-site titrants of serine proteases

Cited by 136 publications

References 0 publications

The pH Dependence of Serpin-Proteinase Complex Dissociation Reveals a Mechanism of Complex Stabilization Involving Inactive and Active Conformational States of the Proteinase Which Are Perturbable by Calcium

The pH Dependence of Serpin-Proteinase Complex Dissociation Reveals a Mechanism of Complex Stabilization Involving Inactive and Active Conformational States of the Proteinase Which Are Perturbable by Calcium

Inactivation of Thrombin by Antithrombin Is Accompanied by Inactivation of Regulatory Exosite I

Analogs of Human Plasminogen That Are Labeled with Fluorescence Probes at the Catalytic Site of the Zymogen

Contact Info

Product

Resources

About