Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator

Abstract

Single-chain urokinase-type plasminogen activator (scu-PA) may be obtained from conditioned cell culture media (natural scu-PA) or by expression of the cDNA encoding human scu-PA in Escheria coli (recombinant scu-PA). The activation of Glu-plasminogen by natural and recombinant scu-PA can be described by a sequence of three reactions, each of which obeys Michaelis-Menten kinetics. Initial activation of plasminogen to plasmin by scu-PA (reaction I) occurs within a high affinity ( K m below 0.8 μM) for both scu-PAs, while the catalytic rate constant ( k 2) is 0.017 s −1 for recombinant scu-PA but only 0.0009 s -1 for natural scu-PA. Subsequent conversion of scu-PA to urokinase (two-chain urokinase-type plasminogen activator, tcu-PA) by generated plasmin (reaction II) occurs with a comparable affinity ( K m about 5 μM) for natural and recombinant scu-PA and with a k 2 of 0.23 s t̄1 for natural and 1.2 s −1 for recombinant scu-PA Finally, activation of plasminogen by tcu-PA (reaction III) occurs with low affinity ( K m 30–50 μM) but with a high catalytic rate constant ( k 2 about 5 s −1) for both natural and recombinant tcu-PA. The differences in the kinetic parameters of the activation of plasminogen by natural and recombinant scu-PA are thus maily due to differences in turnover rate in the first reaction. Indeed, the catalytic rate constant of the first reaction is about 20-times higher for recobinant scu-PA than for natural scu-PA. Thus suprisingly, the artifical, ungloxylated recombinant scu-PA molecule has a better catalytic efficiency glycosated counterpart