| Tissue type plasminogen activator (t-PA) is an unusual serine protease in that its zymogen form, single chain t-PA (sct-PA) is itself an active protease, with approximately 10--25% the enzymatic activity of the mature, two chain form of the enzyme (tct-PA). While much is known about the regulation of t-PA activity within the vasculature as it relates to fibrinolysis, little is known about the regulation of t-PA activity in the central nervous system as it pertains to t-PA's non-vascular roles in neuronal plasticity and neuronal death associated with excitotoxic and ischemic injury. Neuroserpin (NSP), a serine protease inhibitor (serpin), is thought to be the primary cognate inhibitor of t-PA in the extravascular central nervous system, and to inhibit t-PA in a manner analogous to plasminogen activator inhibitor type-1 (PAI-1), the primary inhibitor of t-PA in the vasculature. However, biochemical analysis shows that at pH 7.2 and at 37°C, t-PA inhibition by neuroserpin resembles protease hydrolysis of a substrate with a transient but detectable intermediate more than it resembles the traditional serpin inhibition of a cognate protease via formation of a long-lived acyl-enzyme complex. T-PA forms complexes with NSP and PAI-1 at comparable rates. However, deacylation of NSP-t-PA acyl-enzyme complexes is rapid compared to that of traditional serpin-protease cognate pairs such as PAI-1-t-PA, and is modulated by changes in pH. Interestingly, while at slightly acidic pH the deacylation rates of sct-PA and tct-PA containing acyl-enzyme complexes are equivalent, at pH of 7.4--7.6 tct-PA-NSP complexes deacylate up to five times faster than do sct-PA-NSP complexes.;This thesis presents a kinetic description of the interaction between NSP and both forms of its cognate protease, t-PA, and proposes a novel mechanism for regulating the inhibition of a protease by its cognate serpin. |
