Distinct contributions of residue 192 to the specificity of coagulation and fibrinolytic serine proteases

Archetypal members of the chymotrypsin family of serine proteases, such as trypsin, chymotrypsin, and elastase, exhibit relatively broad substrate spe cificity. However, the successful development of efficient proteolytic casc ades, such as the blood coagulation and fibrinolytic systems, required the evolution of proteases that displayed restricted specificity. Tissue-type p lasminogen activator (t-PA), for example, possesses exquisitely stringent s ubstrate specificity, and the molecular basis of this important biochemical property of t-PA remains obscure. Previous investigations of related serin e proteases, which participate in the blood coagulation cascade, have focus ed attention on the residue that occupies position 192 (chymotrypsin number ing system), which plays a pivotal role in determining both the inhibitor a nd substrate specificity of these enzymes, Consequently, we created and cha racterized the kinetic properties of new variants of t-PA that contained po int mutations at position 192. These studies demonstrated that, unlike in c oagulation serine proteases, Gln-192 does not contribute significantly to t he substrate or inhibitor specificity of t-PA in physiologically relevant r eactions. Replacement of Gln-192 with a glutamic acid residue did, however, decrease the catalytic efficiency of mature, two-chain t-PA toward plasmin ogen in the absence of a fibrin co-factor.