Interaction of streptokinase and rabbit plasminogen

In striking contrast to most other members of the chymotrypsin family of serine proteases, tissue-type plasminogen activator (t-PA) is not synthesized and secreted as a true zymogen. The zymogenicity, or ratio of the catalytic efficiencies of the mature, two-chain enzyme and the single-chain precursor, is only 5-10 for t-PA. This exceptional property of t-PA, however, is not shared by urokinase (u-PA), a plasminogen activator that is very closely related to t-PA. The molecular basis of this important functional distinction between these two intimately related serine proteases has not been previously investigated. Based on observation of the recently described structures of the protease domains of two-chain t-PA and u-PA, and molecular modeling of the corresponding single-chain enzymes, we propose that the presence or absence of an acidic residue at position 144 (chymotrypsin numbering system) is the primary determinant of the distinct zymogenicities of the two enzymes. Consistent with this hypothesis, mutation of histidine 144 of t-PA to an acidic residue, as in u-PA, selectively suppressed the activity of single-chain t-PA and thereby significantly enhanced the enzyme's zymogenicity. A variant of t-PA containing an aspartate residue at position 144, for example, exhibited a zymogenicity of 150, compared to a value of 9 for wild type t-PA and 250 for u-PA.Many critical biological processes depend on specific cleavage of individual target proteins by serine proteases (1-3). One important example is the dissolution of blood clots in which the initiating and rate-limiting step is activation of the circulating zymogen plasminogen (4, 5). In mammalian systems, activation of plasminogen is accomplished by two closely related enzymes, tissue-type plasminogen activator (t-PA) 1 and urokinase (u-PA) (4 -7). t-PA and u-PA possess an extremely high degree of structural similarity (8, 9), share the same primary endogenous substrate and inhibitors (4), and exhibit remarkably stringent substrate specificity (5). In spite of these striking similarities, however, there are clear functional distinctions between the two enzymes. One particularly intriguing distinction is that, by contrast to single-chain u-PA, single-chain t-PA possesses unusually high catalytic activity and is therefore not a true zymogen (10 -13).Proteases are normally synthesized as inactive precursors or zymogens that must either be proteolytically processed or bind to a specific co-factor to develop substantial catalytic activity. The increase in catalytic efficiency after zymogen activation, or zymogenicity, varies widely among individual members of the (chymo)trypsin family but, in almost all cases, is dramatic. For example, strong zymogens such as trypsinogen, chymotrypsinogen, or plasminogen are almost completely inactive with measured zymogenicities of 10 4 to 10 6 (14, 15). Other serine proteases exhibit intermediate zymogenicity. The enzymatic activity of Factor XIIa is 4000-fold greater than that of Factor XII (16), and the catalytic efficiency of ur...

Section: Resultssupporting

confidence: 71%

Converting Tissue-type Plasminogen Activator into a Zymogen

Tachias

Madison

1996

“…Induction of this conformational change in the absence of activation cleavage and concomitant generation of the mature amino terminus is particularly intriguing but not unprecedented. Similar activation of plasminogen upon binding to streptokinase (35)(36)(37)(38)(39)(40) as well as activation of prothrombin by binding to staphylocoagulase (41, 42) have been described previously. Although the mechanism of this nonclassical, nonproteolytic activation of serine protease zymogens remains completely unclear, the behavior of the single-chain variants of t-PA containing mutations in the autolysis loop suggests that Leu 420 , Ser 421 , Pro 422 , and Phe 423 do not play an essential role in this process.…”

Section: Resultssupporting

confidence: 70%

Identification of a Hydrophobic Exosite on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen

Tachias

Lamba

et al. 1997

A wide variety of important biological processes, including both the formation and dissolution of blood clots, depend on specific cleavage of individual target proteins by serine proteases. For example, tissue type plasminogen activator (t-PA), a trypsin-like enzyme that catalyzes the rate-limiting step of the endogenous fibrinolytic cascade, has only one known substrate in vivo, a single peptide bond (Arg 561 -Val 562 ) in the proenzyme plasminogen. We have previously suggested that the specificity of t-PA for plasminogen is mediated in part by direct protein-protein interactions between the protease domain of t-PA and plasminogen that are distinct from those occurring within t-PA's active site. We demonstrate in this study that residues 420 -423 of t-PA, which form a fully solvent-exposed, hydrophobic region of a surface loop mapping near one edge of the active site of t-PA, form, or are essential for the integrity of, an important, secondary site of interaction between t-PA and plasminogen that significantly modulates the rate of plasminogen activation in the absence, but not the presence, of fibrin. Identification of this secondary site of interaction between t-PA and plasminogen provides new insight into molecular details of the evolution of stringent substrate specificity by t-PA and suggests a novel strategy to enhance the fibrin dependence of plasminogen activation by t-PA. While the activity of wild type t-PA is stimulated by fibrin by a factor of approximately 650, the activity of two variants characterized in this study, t-PA/R275E,P422G and t-PA/R275E,P422E, is stimulated by a factor of approximately 39,000 or 61,000, respectively. It is therefore possible that, compared with wild type t-PA, the two variants would display enhanced "clot selectivity" in vivo due to reduced activity in the circulation but full activity at a site of fibrin deposition.Tissue type plasminogen activator (t-PA) 1 has been widely and successfully used as a therapeutic agent to treat acute myocardial infarction (1). t-PA does not, however, dissolve blood clots directly; instead, the enzyme catalyzes conversion of the zymogen plasminogen into the active protease plasmin, which efficiently degrades the fibrin mesh forming the core of a thrombus (2). Both of these critical fibrinolytic enzymes, t-PA and plasmin, are members of the chymotrypsin family of serine proteases (3, 4). Unlike plasmin, however, t-PA is a remarkably specific enzyme. Because such highly restricted substrate specificity is in striking contrast to the broad specificity of well studied serine proteases such as trypsin, chymotrypsin, and elastase (5), the molecular basis of the selectivity of t-PA for plasminogen is of considerable interest. A detailed understanding of the mechanisms employed by t-PA to ensure selectivity would provide new insight into the evolution of the endogenous fibrinolytic cascade and might suggest effective new strategies for the rational design of novel, highly selective proteases with unique specificities as well as novel plasminogen...

“…In addition, through several elegant studies on the mechanism of zymogen activation in the SK⅐HPG complex, it has been shown that HPN exhibits much higher affinity for SK compared with HPG, as a result of which an intermolecular exchange reaction takes place wherein the SK⅐HPN activates free substrate HPG molecules into HPN (10,11). SK can also directly act as a co-factor for HPN, which upon complexation with HPN (12) (Pathway II, direct proteolytic activation pathway) virtually transforms the trypsin-like broad substrate specificity of HPN toward the cleavage of the scissile peptide bond, Arg-561-Val-562, in the incoming substrate HPG (12,13).…”

mentioning

confidence: 99%

Identification through Combinatorial Random and Rational Mutagenesis of a Substrate-interacting Exosite in the γ Domain of Streptokinase

Yadav¹,

Aneja²,

Kumar³

et al. 2011

To identify new structure-function correlations in the ␥ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the ␥ domain and its adjoining region in the ␤ domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the ␥ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the nativelike high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the ␥ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the ␥ domain of streptokinase.Streptokinase (SK), 3 secreted by several ␤-hemolytic group C (1, 2) strains of the genus Streptococcus, is a flexible multidomain protein (3,4). SK is often employed as a thrombolytic drug because it has the ability to activate human plasminogen (HPG), a zymogen, to its enzymatically active form, the serine protease, plasmin (HPN) (5, 6). However, unlike the physiological HPG activators such as tPA and urokinase, SK first forms a high affinity equimolar complex with HPG that results in the conformational activation of HPG to form SK⅐HPG* that specifically recruits substrate HPG and cleaves the Arg-561-Val-562 bond in substrate HPG molecules, thereby converting these into HPN (Pathway I, conformational activation pathway) (5, 6). The N-terminal Ile of SK has been demonstrated to play a critically important role in the activation of the "partner" after the formation of the 1:1 binary complex between SK and HPG (7-9). In addition, through several elegant studies on the mechanism of zymogen activation in the SK⅐HPG complex, it has been shown that HPN exhibits much higher affinity for SK compared with HPG, as a result of which an intermolecular exchange reaction takes place wherein the SK⅐HPN activates free substrate HPG molecules into HPN (10, 11). SK can also directly act as a co-factor for HPN, which upon complexation with HPN (12) (Pathway II, direct proteolytic activation pathway) virtually transforms the trypsin-like broad substrate specificity of HPN toward the cleavage of the scissile peptide bond, Arg-561-Val-562, in the incoming substrate HPG (12, 13).The mechanism by which SK alters the substrate specifi...