Converting Tissue-type Plasminogen Activator into a Zymogen

Abstract

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...

Converting Tissue Type Plasminogen Activator into a Zymogen

“…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 (11,12). Consistent with the latter suggestion, we have demonstrated that conversion of His 144 of t-PA to an acidic residue, which we predicted would alter the conformation of Lys 156 , selectively suppressed the catalytic activity of single-chain t-PA (10).…”

The Conformational Switch from the Factor X Zymogen to Protease State Mediates Exosite Expression and Prothrombinase Assembly

“…This equilibrium lies far to the left when the proteins are unactivated and typically lies far to the right following activation. Notable exceptions are tissue type plasminogen activator, which has significant activity as a zymogen (low zymogenicity), and factor VIIa, which fails to attain its catalytically competent conformation after cleavage of the Arg 15 -Ile 16 bond (high zymogenicity) (53,54). For trypsin and FXa, the zymogen-like conformation is negligibly populated.…”

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.…”