Converting Tissue Type Plasminogen Activator into a Zymogen

Abstract

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 (...

NH2-terminal Structural Motifs in Staphylokinase Required for Plasminogen Activation

“…Substitution of the NH 2 -terminal amino acids by Lys in Sak42D⌬N14 or Sak42D⌬N15, yielding Sak42D⌬N14,A15K and Sak42D⌬N15,S16K, rescued the plasminogen-activating activity, whereas Sak42D⌬N16,Y17K and Sak42D⌬N11,G12K were inactive. Further substitution of Asp 13 and Asp 14 in Sak42D⌬N11,G12K with Asn, yielding Sak42D⌬N11,G12K,D13N,D14N, rescued the plasminogen-activating activity, but a similar substitution of Phe 18 and Glu 19 in Sak42D⌬N16,Y17K with Asn, yielding Sak42D⌬N16,Y17K, F18N,E19N did not endow the latter variant with plasminogen-activating potential. Finally, all variants studied had similar association and dissociation rate constants for low affinity binding to insolubilized native plasminogen and for high affinity binding to insolubilized active site-substituted recombinant plasminogen.…”

“…Recent data concerning the molecular interactions modulating the catalytic efficiency and specificity of serine proteinases support this assumption. Thus, a Lys residue in single-chain tissue plasminogen activator stabilizes the active conformation of this enzyme (19). Moreover, in a series of proteases, Lys may substitute His as the general base in the catalytic triad (20 -22).…”

“…In the present study, the NH 2 -terminal structural elements of staphylokinase required to confer plasminogen-activator specificity to its complex with plasmin were studied by deletion and substitution of amino acids extending from Gly 12 to Glu 19 . These deletion mutants lack plasminogen-activating potential but (some of them) can be functionally rescued by substituting their NH 2 -terminal amino acid with a positively charged Lys, notwithstanding the fact that the Escherichia coli expression system no longer removed the NH 2 -terminal initiation Met from the Lyscontaining variants.…”

Fibrils Colocalize Caspase-3 with Procaspase-3 to Foster Maturation

“…Interestingly, different proteases are locked in this inactive or zymogen state to variable extents (58,68). Table 4 summarizes the zymogenicity values for some well characterized proteases.…”

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

“…Expression of Enzymes by Transient Transfection of COS Cells-cDNAs encoding t-PA; t-PA/R15E; t-PA/Q192E; t-PA/Q192M; t-PA/R15E,Q192E; and t-PA/R15E,Q192M were ligated into the transient expression vector pSVT7 (28) and then introduced into COS cells by electroporation using a Bio-Rad gene pulser as described previously (29,30). 20 g of cDNA, 100 g of carrier DNA, and approximately 10 7 COS cells were placed into a 0.4-cm cuvette, and electroporation was performed at 320 V, 960 microfarads, and ohm ϭ ϱ.…”