(Received for publication, January 9, 1995)
From the
Tissue-type plasminogen activator (t-PA) is a remarkably
specific protease: the only known substrate of this enzyme in vivo is a single peptide bond (Arg-Val
)
within the proenzyme plasminogen. Part of the substrate specificity of
t-PA is due to a ternary interaction between fibrin, t-PA, and
plasminogen which reduces the K
of t-PA
for plasminogen by a factor of 440. However, even in the absence of
fibrin, t-PA continues to hydrolyze plasminogen more rapidly than does
trypsin, a homologous serine protease. We have measured the extent of
the specificity of t-PA for plasminogen by assaying t-PA and trypsin
toward substrates modeled after the peptide sequence in plasminogen
surrounding Arg
-Val
. Surprisingly, t-PA
hydrolyzes these substrates with k
/K
values which
are 28,000-210,000-fold lower than those obtained using trypsin.
Both the high activity toward plasminogen and the low activity toward
peptides are also exhibited by the isolated protease domain. This
suggests that the protease domain, in spite of its high homology to the
nonspecific enzyme trypsin, is inherently specific for recognition of
one or more structural features displayed by native plasminogen.
Tissue type plasminogen activator (t-PA) ()has been
widely and successfully used as a therapeutic agent for the treatment
of acute myocardial infarction(1) . t-PA does not, however,
dissolve blood clots directly; rather, 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.
Both of these important fibrinolytic enzymes, t-PA and plasmin, are
chymotrypsin-like serine proteases(2, 3) . Unlike
plasmin, however, t-PA is a remarkably specific enzyme, a single
peptide bond of plasminogen (Arg
-Val
) is
the only known substrate of t-PA in vivo. 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, the molecular basis of the selectivity of
t-PA for plasminogen is of considerable interest. Moreover, a detailed
understanding of the molecular basis of the extraordinary specificity
of t-PA may facilitate eventual development of the ability to tailor
proteases with desired substrate and inhibitor specificity, a
capability that is very likely to have wide ranging applications.
Mature t-PA is a complex protein that contains 527 amino acids and
is organized by 16 disulfide bonds into five distinct structural
domains that are homologous to similar domains found in other secreted
and cell surface proteins(2, 4, 5) . Residues
4-50 of t-PA form a ``finger'' domain that is closely
related to the fibrin binding finger structures of
fibronectin(6) . Residues 51-87, the ``growth factor
domain,'' share homology with the precursor of epidermal growth
factor (7) and with similar domains in a variety of other
proteins including urokinase (8) , protein C(9) , and
the receptor for low density lipoprotein(10) . Residues
88-175 and 176-263 form two sequential
``kringle'' domains, each built around three characteristic
intradomain disulfide bonds(11, 12, 13) .
Kringle domains are found in a number of other proteins including
plasminogen, thrombin, urokinase, and lipoprotein
(a)(5, 14, 15) . The remainder of the protein
(residues 276-527) constitutes the catalytic domain which shares
homology with other chymotrypsin-like enzymes and contains a typical
catalytic triad (Asp, His
, and
Ser
)(2) .
The chymotrypsin family of serine proteases has evolved to contain many members functioning in the same milieu (e.g. human plasma) that are exclusive in their reactivity toward both substrates and inhibitors. While the role of the ``specificity pocket'' of a serine protease in determining the ``primary'' or P1 specificity of the enzyme has long been appreciated(16, 17, 18, 19) , additional determinants of protease specificity remain largely obscure. Although both the location and mechanism of action of these additional determinants of enzyme specificity remain unclear, their existence and critical importance are well established. For example, t-PA and almost all other proteases that participate in the coagulation cascade, the fibrinolytic cascade, and complement activation possess virtually identical, trypsin-like primary specificity pockets and exhibit identical P1 specificity (i.e. they cleave COOH-terminal to arginine and lysine residues). Nevertheless, most of these enzymes display very different substrate and inhibitor specificity. Previous work from a number of laboratories has shown that, in the absence of fibrin, the isolated protease domain of t-PA is as active as t-PA for cleavage of plasminogen(20, 21, 22, 23, 24) , and a study by Ganu and Shaw (25) suggested that t-PA was inactive toward peptides modeled after the cleavage sequence in plasminogen. We report here the quantitation of the fibrin-independent specificity of t-PA for plasminogen by comparison of the activities of t-PA, t-PA protease domain, and trypsin toward plasminogen and toward linear and cyclic peptides containing the P6-P8` sequence of plasminogen. In contrast to trypsin, t-PA and its isolated protease domain show remarkable specificity for its target sequence in native plasminogen and very little activity toward the related small peptides. The localization and characterization of important determinants of this specificity is essential for understanding the evolution of t-PA as a highly specific protease.
Two properties of t-PA greatly
simplified analysis of data resulting from the indirect assays: (i)
t-PA cleaves plasminogen at only one position
(Arg-Val
) and (ii) unlike plasmin, t-PA
does not hydrolyze Spectrozyme PL. Trypsin shares neither of these
properties with t-PA, complicating the application of this method to
hydrolysis of plasminogen by trypsin. As a result of this limitation,
direct comparison of plasminogen hydrolysis by trypsin and t-PA was
performed by SDS-PAGE analysis of the cleavage products (Fig. 1). Amino-terminal sequencing of the isolated polypeptide
products of these reactions confirmed that the observed cleavage of
plasminogen by both t-PA and trypsin occurred between Arg
and Val
. These experiments also indicated that t-PA (Fig. 1, lanes 8-13) was a more efficient
activator of plasminogen than trypsin (Fig. 1, lanes
2-7). For example, during incubation of 3.3 µM plasminogen with 7.65 nM t-PA or trypsin for 4 h, t-PA
converted almost all the plasminogen (>95%) into plasmin while
trypsin activated <10% of the plasminogen. Additional experiments of
this type were performed, and kinetic parameters for cleavage of
plasminogen by trypsin were evaluated by densitometry of the bands on
SDS-PAGE gels corresponding to residual plasminogen and cleaved
product. The calculated K
and k
values were very similar to those previously reported by Robbins
and co-workers (35) and confirmed that the catalytic efficiency
of cleavage, k
/K
, of
plasminogen by trypsin was lower than for t-PA (Table 1). The
observed 2.4-4.9-fold lower efficiency was entirely the result of
a 9-19-fold reduction in k
, as trypsin
hydrolysis of plasminogen actually exhibited a 4-fold lower K
for plasminogen than did t-PA.
Figure 1: Cleavage of plasminogen by trypsin or tissue plasminogen activator examined by SDS-PAGE. Lane 1 contains uncut plasminogen. Lanes 2-7 contain digests of 3300 nM plasminogen with 7.7 nM trypsin at 20, 40, 60, 120, 180, and 240 min. Lanes 8-13 contain digests of 3300 nM plasminogen with 7.7 nM t-PA.
Figure 2: HPLC analysis of partial tryptic digests of linear and cyclic peptides. The organic solvent gradient rose from 0% at 3 min to 100% at 13.5 min and returned to 0% by 16 min. The traces shown extend from 8 to 16 min. Traces prior to 8 min contained no relevant information and were omitted to remove a peak produced by the N,N-dimethyl formamide in the peptide stock solution of the cyclic peptide. Total amount of peptide loaded was 80 (cyclic) and 40 nmol (linear). Identities of each peak are as labeled.
Trypsin
catalyzed the hydrolysis of both (1) and (2) with
relatively low k values and relatively high K
values as compared to those noted for other
peptide substrates of similar lengths(25) . However, even
though the catalytic efficiency of trypsin toward (1) and (2) was relatively low, its hydrolysis of these substrates was
28,000-210,000-fold more efficient than that of t-PA. This was
reflected both in k
, which was
5,000-51,000-fold lower for t-PA than for trypsin, and in K
, which was 4.1-5.4-fold higher. The
protease domain of t-PA exhibited similarly low catalytic efficiencies
toward (1) and (2) relative to trypsin, and the addition
of fibrin did not accelerate the hydrolysis of either extended peptide
substrate (1) or (2) by t-PA or the protease domain of
t-PA. The introduction of a disulfide in substrate (1) increased
hydrolysis relative to (2), but only by 4-12-fold.
By
contrast to studies with trypsin, kinetic analysis of the cleavage of
peptide substrates by thrombin demonstrated a significant role for
residues at P9 and P10 which are relatively distant in the primary
structure from the scissile bond(39, 40) . To test the
hypothesis that a similar requirement by t-PA could account for the low
activity of t-PA toward peptide (1) (P8-P6`), we synthesized a
24-residue peptide consisting of the P13-P11` residues of plasminogen.
Hydrolysis of the extended peptide by t-PA, however, occurred at a
similar catalytic efficiency to that of peptide (1) (k = 0.0012 s
; K
= 5500 µM; k
/K
= 0.22 M
s
). The molecular
basis of specificity toward peptide substrates appears, therefore, to
differ in important ways among trypsin, thrombin, and t-PA.
Several investigators have reported that the catalytic efficiency toward plasminogen of t-PA and of the isolated protease domain of t-PA are very similar in the absence of fibrin(20, 21, 22, 23, 24) . Important determinants of the fibrin-independent catalysis of plasminogen by t-PA, therefore, appear to map exclusively to the protease domain. Assays of t-PA and the isolated protease domain of t-PA reported here confirm these previous studies. In contrast to the maintenance of high activity toward plasminogen, our assays indicate that the activity of t-PA and isolated protease domain is strikingly lower than trypsin for peptides containing the P6-P8` target sequence in plasminogen. These results extend earlier work by Ganu and Shaw (25) which suggested that t-PA had no activity toward peptides modeled after the cleavage sequence in plasminogen. However, in contrast to this earlier study, we do observe catalysis of peptides by t-PA. The catalytic efficiencies of t-PA toward these peptide substrates were comparable to those reported for variants of trypsin that lack one member of the catalytic triad, suggesting the possibility that, as the free enzyme exists in solution, catalytic and/or substrate binding structures in t-PA are not properly formed or aligned to perform the energetically demanding task of cleaving normal peptide bonds. As indicated in Table 3, however, t-PA and the protease domain of t-PA are able to hydrolyze the more labile amide bonds of p-nitroanilide-containing substrates with kinetic parameters similar to those of trypsin, suggesting that there is no gross disruption of the catalytic machinery.
Comparison of data in Table 1and Table 2reveals that, even in the absence of
fibrin, t-PA is approximately 19,000-87,000 times more active
toward native Lys-plasminogen than toward peptides containing the
cleavage site from plasminogen. In the presence of fibrin this ratio
increases to values varying from 8 10
to 3.6
10
. Data in Table 1and Fig. 1also
demonstrate that the protease domain of t-PA is approximately 3-fold
more catalytically efficient toward plasminogen than is trypsin and
that trypsin is 29,000-550,000 times more efficient than the
protease domain of t-PA for catalysis of the same cleavage sequence
within short peptides. The structural context of the labile amide
linkage, therefore, results in a 87,000-1,650,000-fold difference
in the relative reactivities of trypsin and the protease domain of t-PA
toward the same primary structure. While proper folding of a substrate
is a hindrance to most proteases, it seems to be required for efficient
cleavage by t-PA. The equally low activities of t-PA and the isolated
protease domain excludes the possibility that the low activity toward
peptides was due to the inhibition of the protease domain by one of the
non-protease domains. Conversely, the similarity in the
fibrin-independent activities of t-PA and the isolated protease domain
toward plasminogen indicates that the non-protease domains do not
substantially contribute to the fibrin-independent activation of native
plasminogen by t-PA.
The observation that t-PA and the protease
domain of t-PA possess similarly high activity toward native
plasminogen suggests that, in the absence of fibrin, important
interactions between t-PA and plasminogen require only the protease
domain of t-PA. In the absence of structural information for either
t-PA or plasminogen, understanding of the molecular basis of the
substrate specificity of t-PA will necessarily remain inexact. However,
the finding that t-PA processes both linear and cyclic peptide mimics
of its target sequence extremely inefficiently does suggest that t-PA
recognizes complex and/or multiple elements on the surface of native
plasminogen whose precise conformation or alignment is likely to be
dependent on the tertiary structure of plasminogen. One possible
explanation of this observation is that binding of plasminogen induces
a subtle alteration in the catalytic machinery of the protease domain
of t-PA which enables it to efficiently cleave peptide amide bonds.
Alternatively, some aspect of the secondary or tertiary structure of
plasminogen may be required to interact with the protease domain to
correctly align the labile Arg-Val
bond for
cleavage.
The recent description of the structure of complement Factor D provides the first evidence that the former mechanism might be utilized by highly specific proteases to restrict substrate specificity. Examination of the 2.0-Å crystal structure of complement Factor D reveals that alterations relative to trypsin at residues 94, 214, and 215 prevent aspartic acid 102 from interacting with histidine 57 in the free enzyme. Narayana and co-workers (43) propose, therefore, that binding of the substrate (a complex of Factor B and the co-factor C3b) to Factor D must cause the catalytic triad and the S1 pocket of Factor D to assume an active conformation and thereby promote rapid catalysis of the target peptide bond. While t-PA and complement Factor D both possess high reactivity toward their specific substrate and low reactivity toward peptides, complement Factor D is functionally different from t-PA because it is relatively unreactive toward ester and activated amide substrates. This functional distinction between the two proteases suggests that the observed disruption of the catalytic machinery of complement Factor D is probably more severe than any disruption which may be present in t-PA. Consequently, the structural basis for the low reactivity of the two proteases toward peptide substrates is not likely to be identical, suggesting that multiple mechanisms may have evolved to enhance the specificity of serine proteases.
We have demonstrated that, even in the absence of fibrin, t-PA is more active than trypsin toward plasminogen and that this efficient catalysis is in contrast to the dramatically lower catalysis by t-PA of other peptide and protein substrates. We also provide the first quantitative treatment of the cleavage by t-PA of linear and cyclic peptides that contain its normal target sequence from plasminogen. These assays indicate that t-PA is 19,000-87,000 times more active toward plasminogen than toward peptides containing the cleavage site from plasminogen. Important determinants of this impressive specificity for cleavage of native plasminogen appear to reside in the protease domain of t-PA. Kinetic measurements reported in this study emphasize, and also quantify, profound differences between the protease domain of t-PA and trypsin, two highly homologous enzymes. This study may facilitate the identification of peptides that mimic the ``recognition surface'' on plasminogen and offers an incentive for homology modeling of the protease domain of t-PA based upon the structure of trypsin. Such efforts may provide important new insights regarding the evolution of the protease domain of t-PA into an exquisitely specific protease and may facilitate the development of more selective thrombolytic agents.