Reaction of human chymase with reactive site variants of alpha 1-antichymotrypsin. Modulation of inhibitor versus substrate properties.

Schechter, Norman M.; Jordan, Lynda; James, A. M.; Cooperman, Barry S.; Wang, Zhi Mei; Rubin, Harvey

doi:10.1016/s0021-9258(19)49508-2

Cited by 57 publications

(3 citation statements)

References 30 publications

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“…of aj-PI upon changing the PI4 residue to arginine without an alteration in the underlying mechanism of interaction with proteinase are in agreement with a number of recent studies on related serpins, which have been interpreted in terms of a common branched pathway, suicide substrate inhibition mechanism, with perturbation of fc4 and/or k3 resulting in sometimes large changes in S.I. (i) Rubin and colleagues have shown that mutation of residues within the reactive center loop of «iantichymotrypsin altered the stoichiometry of inhibition toward different proteinases as a result of alteration in 1k4 and/or k3 (Rubin et al, 1990;Schechter et al, 1993). (ii) A P10 hinge region mutant of ai-PI, in which glycine was substituted by proline, reacted with both trypsin and HNE predominantly as a substrate to give a more stable loop-inserted form (Hopkins et al, 1993) but continued to function as an inhibitor, albeit an inefficient one.…”

Section: Discussionsupporting

confidence: 87%

.alpha.1-Proteinase Inhibitor Variant T345R. Influence of P14 Residue on Substrate and Inhibitory Pathways

Hood¹,

Huntington²,

Gettins³

1994

Biochemistry

109

104

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To test whether the presence of charged residues at position P14 of the reactive center region of noninhibitory members of the serpin family of protein proteinase inhibitors is responsible for their lack of proteinase inhibitory properties, we expressed a variant of the alpha 1-proteinase inhibitor (alpha 1-PI) with arginine substituted for threonine at this position (T345R) and characterized its functional properties. Although the T345R variant reacted with proteinases principally as a substrate, it was still capable of forming stable complexes with the three serine proteinases examined, human neutrophil elastase (HNE), porcine pancreatic elastase (PPE), and trypsin. The fraction of T345R alpha 1-PI that formed a complex with proteinase was quantitated by autoradiography of SDS gels of the variant incubated with 125I-labeled proteinase. The stoichiometry of inhibition (S.I.) (number of mol of alpha 1-PI required to completely inhibit 1 mol of proteinase), which was 1 for both plasma alpha 1-PI and wild-type recombinant alpha 1-PI interacting with each of the proteinases, was very much greater than 1 for T345R variant alpha 1-PI. Values of 9.5, 45, and about 70 were estimated for variant alpha 1-PI inhibition of trypsin, HNE, and PPE, respectively. An inverse relationship between the apparent second-order rate constant and the S.I. for inhibition of PPE by T345R alpha 1-PI suggested that the mutation did not affect the rate-determining step of formation of a transient intermediate complex. Following cleavage of the reactive center loop, there was a large increase in protein stability and changes in the CD spectrum, both consistent with insertion of the reactive center loop into beta-sheet A. This behavior is similar to that of wild-type alpha 1-PI. We conclude that the presence of a charged residue at P14 does not prevent reactive center loop insertion or the functioning of alpha 1-PI as an inhibitor of serine proteinases but does significantly alter the relative rates of the substrate and inhibitory pathways in favor of the former, probably by reducing the rate of the latter reaction.

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Section: Discussionsupporting

confidence: 87%

.alpha.1-Proteinase Inhibitor Variant T345R. Influence of P14 Residue on Substrate and Inhibitory Pathways

Hood¹,

Huntington²,

Gettins³

1994

Biochemistry

109

104

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“…When analyzed by SDS−PAGE, serpin−protease complexes typically migrate at a size equivalent to the sum of the MWs of the protease and serpin, implying the covalent linkage. In keeping with a suicide substrate, the trapping mechanism is not always 100% efficient, and SDS−PAGE of protease-serpin reactions often shows a band of hydrolyzed-inactivated serpin at a slightly lower MW position than the intact serpin ( , ). Thus, serpins are expected to react only with dissociated forms of HTβ that have catalytic ability.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Three Distinct Catalytic Forms of Human Tryptase-β: Their Interrelationships and Relevance

et al. 2007

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Human tryptase-beta (HTbeta) is a serine protease that is isolated as a tetramer of four identical, catalytically active subunits (HTbeta-AT). Tetramer activity is not affected by protein-based physiological inhibitors but instead may be regulated by an autoinactivation process we have called spontaneous inactivation. Unless stabilized by heparin or high salt, the active tetramer converts to an inactive state consisting of an inactive-destabilized tetramer that reversibly dissociates to inactive monomers upon dilution. We refer to this mixture of inactive species as siHTbeta and show in this study that previous reports of monomeric catalytic forms are derived from this mixture. siHTbeta itself did not hydrolyze model substrates but unlike the tetramer did react slowly with the serpin alpha2-antiplasmin (alpha2-AP), suggesting a highly limited catalytic potential. In the presence of heparin (or other highly charged polysaccharides), we demonstrate that siHTbeta formed a well-defined complex with the heparin (siHTbeta-HC) that reacted 70-fold faster with alpha2-AP than siHTbeta and also hydrolyzed model substrates and fibrinogen. Formation of siHTbeta-HC was limited to dilute subunit solutions since high subunit concentrations resulted in the reformation of the active tetramer. By compensating for changes in the strength of heparin binding, siHTbeta-HC could be formed over the pH range of 6.0-8.5. The activity dependence on pH was bell-shaped with highest activity between pH 6.8 and pH 7.5. In contrast, HTbeta-AT activity showed no dependence upon heparin, increased over the pH range of 6.0-8.5, and was much higher than that of siHTbeta-HC especially above pH 6.8. HTbeta-AT incubated with excess heparin of different size (3-15 kDa) was functionally stable at 25 degrees C but lost activity regardless of heparin size at 37 degrees C above pH 6.8. The change in stability, which is likely due to weakened heparin binding, did not result in the formation of a stable catalytic monomer. These results confirm that siHTbeta is for the most part an inactive species and that any active monomer is a consequence of heparin binding to siHTbeta under dilute conditions where unfavorable thermodynamics and/or kinetics restrict formation of active tetramer. Heparin binding under these conditions drives a limited reorganization of the active site to a conformation that is catalytic but not the equivalent of a subunit within the active tetramer.

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“…These changes also altered the proteases' preferences for P3 and P3' residues in peptide substrates and activities with physiological substrates. Mutations in serpins have focused on the reactive site loop residues, primarily P2, PI, and PT (Derechin et al, 1990;Eldering et al, 1992;Heeb et al, 1990;Lane et al, 1991;Schechter et al, 1993), and on residues P10-P14, which partially insert into the /3-sheets of the body of the serpin (Hopkins et al, 1993;Lane et al, 1991). These variant proteins have demonstrated the remarkable specificity of most enzymes for their substrates; however, individual residues in contact between the protease and serpin have not been identified.…”

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confidence: 99%

Intermolecular Interactions between Protein C Inhibitor and Coagulation Proteases

Cooper

Whinna

Jackson³

et al. 1995

Biochemistry

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Protein C inhibitor (PCI) inhibits multiple plasma serine proteases. To determine which residues contribute to its specificity of inhibition, 19 mutations in the reactive site loop of PCI (from Thr352 to Arg357) were generated and assayed with thrombin, activated protein C (APC), and factor Xa. To identify the intermolecular interactions responsible for these kinetics, a molecular model of PCI was generated using alpha 1-protease inhibitor and ovalbumin as templates. This model of PCI was docked with thrombin, followed by extensive energy minimization, to determine a lowest energy complex. The resulting docked complex was used as a template to form molecular models of PCI-APC and PCI-factor Xa complexes. The best inhibitors of thrombin contained Pro or Gly at the P2 position in place of Phe353, with 2- and 7-fold increases in activity, respectively. These substitutions reduced steric interactions with the 60-insertion loop unique to thrombin. The best inhibitors of APC and factor Xa contained Arg at the P3 position in place of Thr352, with 2- and 5-fold increases in inhibition rates, respectively. The molecular model predicts that Arg in this position could form a salt bridge with Glu217 of each protease. Changing Arg357 at the P3' position had little effect on protease inhibition, consistent with the observation in the model that this residue points toward the body of PCI, forming a salt bridge with Glu220. Given its broad specificity of inhibition, PCI has proven very useful in understanding the nature of serpin-protease interactions using multiple mutations in a serpin assayed with multiple proteases.

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Reaction of human chymase with reactive site variants of alpha 1-antichymotrypsin. Modulation of inhibitor versus substrate properties.

Cited by 57 publications

References 30 publications

.alpha.1-Proteinase Inhibitor Variant T345R. Influence of P14 Residue on Substrate and Inhibitory Pathways

.alpha.1-Proteinase Inhibitor Variant T345R. Influence of P14 Residue on Substrate and Inhibitory Pathways

Characterization of Three Distinct Catalytic Forms of Human Tryptase-β: Their Interrelationships and Relevance

Intermolecular Interactions between Protein C Inhibitor and Coagulation Proteases

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