Extended Mechanism and Rate-Limiting Step of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis

28 October 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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 based on simplifying approximations may be inaccurate for complex mechanisms like that of staphylokinase. We employed a modern numerical approach of global kinetic data analysis whereby steady-state kinetics and binding affinity datasets 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 10,000-fold higher than that reported in the literature. The model further showed that the rate-limiting step of the catalytic cycle is the 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, we 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. Our study provides an interesting example of how global numerical analysis of kinetic data can be used to better understand the mechanism and limiting factors of complex biochemical processes.

Keywords

staphylokinase
global kinetic analysis
mechanism
rate limitation
cardiovascular diseases
thrombolytics

Supplementary materials

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SUPPORTING INFORMATION
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Materials and Methods. Supplementary Figures and Tables.
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