Variants of Tissue-type Plasminogen Activator That Display Extraordinary Resistance to Inhibition by the Serpin Plasminogen Activator Inhibitor Type 1

Tachias, Kathy; Madison, Edwin L.

doi:10.1074/jbc.272.23.14580

Cited by 22 publications

(14 citation statements)

References 51 publications

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“…5 It would be consistent with the higher k cat reported for chromogenic substrate hydrolysis by two-chain tPA than that for hydrolysis by the single-chain form (37). The primary effect of the exosite interactions associated with complementary charged amino acid residue side chains in tPA and PAI-1 should be to increase k 1 , an assumption consistent with the demonstration that mutant forms of tPA which lack one or several of the positively charged residues in the 37-loop exhibit reduced rates of inhibition by PAI-1 (17). If the exosite bonds remain intact in the Michaelis complexsand this should apply at least to the bond between the P4′ Glu and Arg39 in tPAsthey will hamper the reactivity of the Michaelis complex in all directions that depend on breaking the exosite bonds, such as forming a stabilized acyl enzyme complex through loop insertion and a concomitant translocation of the proteinase.…”

Section: Discussionmentioning

confidence: 69%

Inhibitory Mechanism of Serpins: Loop Insertion Forces Acylation of Plasminogen Activator by Plasminogen Activator Inhibitor-1

Kvassman¹,

Verhamme²,

Shore³

1998

Biochemistry

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Serpin inhibitors are believed to form an acyl enzyme intermediate with their target proteinases which is stabilized through insertion of the enzyme-linked part of the reactive center loop (RCL) as strand 4 in beta-sheet A of the inhibitor. To test critically the role and timing of these steps in the reaction of the plasminogen activator inhibitor PAI-1, we blocked the vacant position 4 in beta-sheet A of this serpin with an octapeptide. The peptide-blocked PAI-1 was a substrate for both tissue-type plasminogen activator (tPA) and trypsin and was hydrolyzed at the scissile bond. The reactivity of the peptide-blocked substrate PAI-1 was compared to that of the unmodified inhibitor by rapid acid quenching as well as photometric techniques. With trypsin as target, the limiting rate constants for enzyme acylation were essentially the same with inhibitor and substrate PAI-1 (21-23 s-1), as were also the associated apparent second-order rate constants (2.8-2.9 microM-1 s-1). With tPA, inhibitor and substrate PAI-1 reacted identically to form a tightly bound Michaelis complex (Kd approximately Km approximately 20 nM). The limiting rate constant for acylation of tPA, however, was 57 times faster with inhibitor PAI-1 (3.3 s-1) than with the substrate form (0.059 s-1), resulting in a 5-fold difference in the corresponding second-order rate constants (13 vs 2.5 microM-1 s-1). We attribute the ability of tPA to discriminate between the two PAI-1 forms to exosite bonds that cannot occur with trypsin. The exosite bonds retain specifically the distal part of the PAI-1 RCL in the substrate pocket, which favors a reversal of the acylation step. Acylation of tPA becomes effective only by separating the products of the acylation step. With substrate PAI-1, this depends on passive displacement of bonds, whereas with inhibitor PAI-1, separation is accomplished by loop insertion that pulls tPA from its docking site on PAI-1, resulting in faster acylation than with substrate PAI-1.

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Section: Discussionmentioning

confidence: 69%

Inhibitory Mechanism of Serpins: Loop Insertion Forces Acylation of Plasminogen Activator by Plasminogen Activator Inhibitor-1

Kvassman¹,

Verhamme²,

Shore³

1998

Biochemistry

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“…Measurement of Second Order Rate Constants for Inhibition by PAI-1-Second order rate constants (k i ) for the inhibition of wild type and mutated t-PA were measured under pseudo-first order conditions as described previously (32)(33)(34)(35)(36). Inhibitor was present in at least a 20-fold molar excess over enzyme.…”

Section: Conversion Of Single-chain T-pa Preparations Into Two-chain mentioning

confidence: 99%

Distinct Contributions of Residue 192 to the Specificity of Coagulation and Fibrinolytic Serine Proteases

Zhang

Hervio

Strandberg

et al. 1999

Journal of Biological Chemistry

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Archetypal members of the chymotrypsin family of serine proteases, such as trypsin, chymotrypsin, and elastase, exhibit relatively broad substrate specificity. However, the successful development of efficient proteolytic cascades, such as the blood coagulation and fibrinolytic systems, required the evolution of proteases that displayed restricted specificity. Tissue-type plasminogen activator (t-PA), for example, possesses exquisitely stringent substrate specificity, and the molecular basis of this important biochemical property of t-PA remains obscure. Previous investigations of related serine proteases, which participate in the blood coagulation cascade, have focused attention on the residue that occupies position 192 (chymotrypsin numbering system), which plays a pivotal role in determining both the inhibitor and substrate specificity of these enzymes. Consequently, we created and characterized the kinetic properties of new variants of t-PA that contained point mutations at position 192. These studies demonstrated that, unlike in coagulation serine proteases, Gln-192 does not contribute significantly to the substrate or inhibitor specificity of t-PA in physiologically relevant reactions. Replacement of Gln-192 with a glutamic acid residue did, however, decrease the catalytic efficiency of mature, two-chain t-PA toward plasminogen in the absence of a fibrin co-factor.Appreciation that thrombotic disorders are the major cause of morbidity and mortality in many countries sparked intense interest in the human fibrinolytic system, which normally provides a counterbalance to the blood coagulation cascade (1-5). The rate-limiting step in the fibrinolytic cascade, conversion of the circulating zymogen plasminogen into the active protease plasmin, is catalyzed by t-PA, 1 a member of the chymotrypsin family of serine proteases (4, 6, 7). Discovery of this important physiological role encouraged extensive scrutiny of t-PA, and the efforts of many investigators have placed this enzyme among the most thoroughly characterized human enzymes (6 -8). Administration of wild type t-PA has now become standard therapy for acute myocardial infarction (5, 9 -13), and several variants of the enzyme have either recently entered phase III clinical trials for testing as improved thrombolytic agents or already received approval by the Food and Drug Administration (14, 15).One of the most important and intriguing biochemical properties of t-PA is the highly stringent substrate and inhibitor specificity (6, 7), which is in stark contrast to that of archetypal chymotrypsin family enzymes such as chymotrypsin, trypsin, and elastase (16). The striking substrate specificity of t-PA is mediated in part by interactions of both the enzyme and its substrate with the co-factor fibrin (6, 7). However, even in the absence of a co-factor, t-PA maintains strict specificity for plasminogen, the primary physiological substrate, and we have shown that this specificity is an inherent property of the isolated protease domain of t-PA (17). Recent structural...

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“…Unique to the PAI-1 inhibitory mechanism are the exosite interactions between the surface-exposed variable region-1 (VR1) or 37-loop of tPA and the distal RCL of PAI-1 (13,14). These interactions are believed to facilitate the rate of formation of Michaelis complex (tPA⅐PAI-1), although direct evidence for this hypothesis is still lacking (15)(16)(17).…”

mentioning

confidence: 99%

The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

Ibarra¹,

Blouse

Christian³

et al. 2004

Journal of Biological Chemistry

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The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and ␤-trypsin by PAI-1. Alanine substitutions at the distal P4 (Glu-350) and P5 (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k a ) and overall inhibition (k app ) with tPA by 13.4-and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k lim ) increased by 3.3-fold. The effects of double mutations on k a , k lim , and k app were small with uPA and nonexistent with ␤-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into ␤-sheet A. Moreover, mutational analysis indicates that the P5 residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.Plasminogen activator inhibitor-1 (PAI-1) 1 functions as the primary regulator of the fibrinolytic system by inhibiting the conversion of plasminogen into plasmin via its action on tissuetype plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (1). This inhibitor also plays a vital role in other physiological processes, including tumor invasion and tissue remodeling (2). PAI-1 belongs to the serpin superfamily, which shares several unique structural features, including a five-stranded ␤-sheet motif and a flexible reactive center loop (RCL). The conformational changes associated with these structures have been linked to the inhibitory function of PAI-1 (3-9). Notably, the mechanism of inhibition depends on the structural changes accompanying the S (stressed) to R (relaxed) transition that results in complete insertion of the Nterminal (proximal) part of the RCL into ␤-sheet A as an additional ␤-strand, s4A. The proteinase, tethered by a covalent bond with the P1 residue of the serpin, is displaced from the initial docking site to the opposite end of the serpin molecule, separating the P1Ј and P1 residues by ϳ70 Å (10 -12). This mechanism efficiently distorts and inactivates the catalytic triad of the proteinase, stabilizing the serpin-proteinase complex at the acyl-enzyme intermediate stage.Unique to the PAI-1 inhibitory mechanism are the exosite i...

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Variants of Tissue-type Plasminogen Activator That Display Extraordinary Resistance to Inhibition by the Serpin Plasminogen Activator Inhibitor Type 1

Cited by 22 publications

References 51 publications

Inhibitory Mechanism of Serpins: Loop Insertion Forces Acylation of Plasminogen Activator by Plasminogen Activator Inhibitor-1

Inhibitory Mechanism of Serpins: Loop Insertion Forces Acylation of Plasminogen Activator by Plasminogen Activator Inhibitor-1

Distinct Contributions of Residue 192 to the Specificity of Coagulation and Fibrinolytic Serine Proteases

The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

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