Abstract
Archetypal members of the chymotrypsin family of serine proteases, such as trypsin, chymotrypsin, and elastase, exhibit relatively broad substrate specificity. However, the successful development of efficient proteolytic cascades, such as the blood coagulation and fibrinolytic systems, required the evolution of proteases that displayed restricted specificity. Tissue-type plasminogen activator (t-PA), for example, possesses exquisitely stringent substrate specificity, and the molecular basis of this important biochemical property of t-PA remains obscure. Previous investigations of related serine proteases, which participate in the blood coagulation cascade, have focused attention on the residue that occupies position 192 (chymotrypsin numbering system), which plays a pivotal role in determining both the inhibitor and substrate specificity of these enzymes. Consequently, we created and characterized the kinetic properties of new variants of t-PA that contained point mutations at position 192. These studies demonstrated that, unlike in coagulation serine proteases, Gln-192 does not contribute significantly to the substrate or inhibitor specificity of t-PA in physiologically relevant reactions. Replacement of Gln-192 with a glutamic acid residue did, however, decrease the catalytic efficiency of mature, two-chain t-PA toward plasminogen in the absence of a fibrin co-factor.
Highlights
Appreciation that thrombotic disorders are the major cause of morbidity and mortality in many countries sparked intense interest in the human fibrinolytic system, which normally provides a counterbalance to the blood coagulation cascade [1,2,3,4,5]
One of the most important and intriguing biochemical properties of type plasminogen activator (t-PA) is the highly stringent substrate and inhibitor specificity [6, 7], which is in stark contrast to that of archetypal chymotrypsin family enzymes such as chymotrypsin, trypsin, and elastase [16]
Rezaie, Morrissey, and their co-workers (20 –25) have reported a number of interesting studies that suggest the residue at position 192 of the coagulation proteases plays a pivotal role in mediating the inhibitor and substrate specificity of these enzymes
Summary
Site-directed Mutagenesis and Construction of Expression Vectors Encoding Variants of t-PA—Oligonucleotide-directed site-specific mutagenesis was performed by the method of Zoller and Smith [26] as modified by Kunkel [27]. Conversion of Single-chain t-PA Preparations into Two-chain Enzymes—The t-PA and t-PA variants produced by COS-1 cells were present predominantly in the single-chain form These single-chain enzymes were converted into the corresponding, mature two-chain enzymes by incubation with plasmin-Sepharose as described previously [31], and quantitative cleavage was verified by polyacrylamide gel electrophoresis followed by Western blotting. Kinetic Analysis of Plasminogen Activation Using Indirect Chromogenic Assays—Indirect chromogenic assays of t-PA utilized the substrates Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica) and were performed as described previously [32, 33]. Data were analyzed by plotting ln(residual activity/initial activity) versus time of preincubation and measuring the resulting slopes Division of this slope by Ϫ[I], with [I] representing the concentration of TABLE I Kinetic constants for cleavage of the chromogenic substrates by singleand two-chain t-PA variants
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