Extended mechanism of staphylokinase (SAK). (a) Simplified graphical schematic of the mechanism and (b) detailed kinetic pathway with all analyzed steps and parameters provided for clarity. In comparison to the general mechanism (Figure 1), the extended mechanism newly includes binding of staphylokinase (SAK) to plasminogen (Plg) to form an inactive complex, preventing plasminogen to plasmin (Plg to Plm) conversion and having a significant effect on the overall effectivity. The mechanism further includes a newly identified two-step induced-fit binding mechanism for both Plm and Plg binding by SAK. SAK·Plg* to SAK·Plm* conversion by Plm, reported previously (15) and marked with a gray dotted arrow, was not included in the kinetic model because its rate was very slow and not significantly detectable during the experimental time window. Forward and reverse rate constants of the ith step are denoted with the symbols k+i and k–i, respectively. The steady-state kinetic parameters Km, kcat, and Kp correspond to the Michaelis constant, turnover number, and product inhibition constant, respectively.
Zbyněk Prokop Research Group
Significance
The plasminogen activator staphylokinase is a fibrin-specific thrombolytic biomolecule and an attractive target for the development of effective myocardial infarction and stroke therapy. To engineer the protein rationally, a detailed understanding of the biochemical mechanism and limiting steps is essential. Conventional fitting to equations derived on the basis of simplifying approximations may be inaccurate for complex mechanisms such as that of staphylokinase. We employed a modern numerical approach of global kinetic data analysis whereby steady-state kinetics and binding affinity data sets were analyzed in parallel. Our approach provided an extended, revised understanding of the staphylokinase mechanism without simplifying approximations and determined the value of turnover number kcat of 117 s–1 that was 10000-fold higher than that reported in the literature. The model further showed that the rate-limiting step of the catalytic cycle is binding of staphylokinase to plasmin molecules, which occurs via an induced-fit mechanism. The overall staphylokinase effectivity is further influenced by the formation of an inactive staphylokinase·plasminogen complex. Here, Z. Prokop et.al. describe a quick and simplified guide for obtaining reliable estimates of key parameters whose determination is critical to fully understand the staphylokinase catalytic functionality and define rational strategies for its engineering. Their study provides an interesting example of how a global numerical analysis of kinetic data can be used to better understand the mechanism and limiting factors of complex biochemical processes. The high catalytic activity of staphylokinase (more than 1000-fold higher than that of the clinically used drug alteplase) determined herein makes this thrombolytic agent a very attractive target for further engineering.
Toul, M., Nikitin, D., Marek, M., Damborsky, J., and Prokop, Z:
Extended Mechanism of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis: 1000-fold Higher Catalytic Activity than That of Clinically Used Alteplase, ACS Catal. 2022, 12, 7, 3807-3814, https://doi.org/10.1021/acscatal.1c05042