Human urokinase type plasminogen activator (u-PA) is a member of the chymotrypsin family of serine proteases that can play important roles in both health and disease. We have used substrate phage display techniques to characterize the specificity of this enzyme in detail and to identify peptides that are cleaved 840 -5300 times more efficiently by u-PA than peptides containing the physiological target sequence of the enzyme. In addition, unlike peptides containing the physiological target sequence, the peptide substrates selected in this study were cleaved as much as 120 times more efficiently by u-PA than by tissue type plasminogen activator (t-PA), an intimately related enzyme. Analysis of the selected peptide substrates strongly suggested that the primary sequence SGRSA, from position P3 to P2, represents optimal subsite occupancy for substrates of u-PA. Insights gained in these investigations were used to design a variant of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both u-PA and t-PA, that inhibited u-PA approximately 70 times more rapidly than it inhibited t-PA. These observations provide a solid foundation for the design of highly selective, high affinity inhibitors of u-PA and, consequently, may facilitate the development of novel therapeutic agents to inhibit the initiation and/or progression of selected human tumors.

In stark 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. Instead, single-chain t-PA exhibits very significant catalytic activity. Consequently, the zymogenicity, or ratio of the catalytic efficiencies of the mature, two-chain enzyme and the single-chain precursor, is only 3-9 for t-PA. Both we and others have previously proposed that Lys 156 may contribute directly to this exceptional property of t-PA by forming interactions that selectively stabilize the active conformation of the single-chain enzyme. To test this hypothesis we created variants of t-PA in which Lys 156 was replaced by a tyrosine residue. As predicted, the K156Y mutation selectively suppressed the activity of the single-chain enzyme and thereby substantially enhanced the enzyme's zymogenicity. In addition, however, this mutation produced a very dramatic increase in the ability of single-chain t-PA to discriminate among distinct fibrin co-factors. Compared with wild type t-PA, one of the variants characterized in this study, t-PA/R15E,K156Y, possessed substantially enhanced response to and selectivity among fibrin co-factors, resistance to inhibition by plasminogen activator inhibitor type 1, and significantly increased zymogenicity. The combination of these properties, and the maintenance of full activity in the presence of fibrin, suggest that the R15E,K156Y mutations may extend the therapeutic range of t-PA.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 (1, 2). Other serine proteases exhibit intermediate zymogenicity. The enzymatic activity of Factor XIIa is 4000-fold greater than that of Factor XII (3), and the catalytic efficiency of urokinase is 250-fold greater than that of prourokinase (4). By contrast, the catalytic activities of single-and two-chain t-PA 1 vary by a factor of only 3-9 (5-9).We have suggested previously that the unusually high catalytic activity of single-chain t-PA results both from the absence of interactions, present in typical zymogens, that stabilize (an) inactive conformation(s) of the zymogen and the presence of interactions, absent in typical zymogens, that stabilize an active conformation of the single-chain enzyme (8 -10). Recent studies have provided substantial support for this hypothesis. We demonstrated that the absence of the zymogen triad contributes to the enzymatic activity of single-chain t-PA (8, 9), and two groups have suggested that Lys 156 2 stabilizes an active conformation of single-chain t-PA (...

The methanol-insoluble, heat-stable enterotoxin of Escherichia coli synthesized by clinical strains or strains that harbor the cloned gene was shown to be an extraceilular polypeptide. The toxin (STB) was first detected as an 8,100-M, precursor (pre-STB) that was converted to a transiently cell-associated 5,200-Mr form.Proteolytic conversion of pre-STB to STB was shown to be inhibited by the proton motive force uncoupler carbonyl cyanide m-chlorophenylhydrazone and did not occur in a secA background. After STB was detected as a cell-associated molecule, an extracellular form with identical electrophoretic mobility became apparent. The results suggest that there is no proteolytic processing during the mobilization of STB from the periplasm to the culture supernatant. The determined amino acid sequence of STB coincides fully with the 48 carboxy-terminal amino acids inferred from the DNA sequence. The 23 amino-terminal residues inferred from the DNA sequence were absent in the mature toxin.Enterotoxigenic Escherichia coli synthesizes the heatlabile and the heat-stable (ST) families of enterotoxins; these toxins have been shown to be responsible for secretory diarrhea in humans and animals (reviewed in references 1 and 30). STs have been classified as methanol soluble (STA) and methanol insoluble (STB) (3, 36), and these subdivisions correlate well with the inferred or known amino acid compositions of the toxins (18,21,27). The toxic activity of STA is resistant to proteases (6, 33), while STB is inactivated upon trypsin treatment (35). STAs are 18-or 19-amino-acid extracellular enterotoxins that result from two independent proteolytic cleavages on a 72-amino-acid precursor (prepro-STA); the first cleavage yields a periplasmic 53-aminoacid pro-STA that is extracellularly processed to mature STA (29a). The three disulfide bridges formed by the six cysteine residues of STA are sine qua non for toxic activity (6,10,33). A structural model based on proton nuclear magnetic resonance has been proposed for this toxin (11,24). It is also known that STA interacts with an enterocyte receptor that activates guanylate cyclase and results in increased intestinal secretion (8); in contrast to STA, very little is known about the mechanism of action and the export-secretion pathway of methanol-insoluble STB; its gene (estB) has been sequenced (21, 27), and the 71-codon open reading frame, when translated, is very different from the 72 residues of the precursor of STA (reviewed in reference 18). The first 23 amino-terminal amino acids inferred from the estB sequence have properties compatible with a signal peptide (21, 27), and it has been proposed that mature STB is a 48-residue molecule; it was unclear, however, whether STB is an extracellular polypeptide secreted into the medium like STA (16) or whether it is a periplasmic enterotoxin like heat-labile enterotoxin (26). In this communication, we show that mature STB is a 48-amino-acid extracellular polypeptide that corresponds to the previously inferred carboxy-terminal * Correspo...

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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...