Barbara Saccà, ZMB, University of Duisburg-Essen, Essen, Germany

The effect of DNA boundaries on the kinetic linkage of a monomeric serine protease

Allosterically regulated enzymes are characterized by a non-Michaelis Menten kinetics described by a non-hyperbolic relationship between the initial reaction rate and substrate concentration. Whereas in oligomeric proteins such thermodynamic cooperativity has been explained in terms of mechanically coupled site-to-site interactions at equilibrium conditions, emergence of this phenomenon in monomeric enzymes has been more rarely observed and is essentially of kinetic origin. Inspired by the increased interest in DNA-origami/protein complexes and the still open questions on their kinetic properties, we investigated the impact of unspecific electrostatic interactions and programmable spatial confinement on the non Michaelis-Menten mechanism of thrombin, a paradigm of monomeric serine proteases. The thrombin-catalyzed proteolysis of distinct substrates was analyzed either in presence or absence of thrombin-binding aptamers, with these latter occurring in solution either as free unbound species or tethered to the inner walls of DNA origami cages of different shape and structural flexibility. In our hands, the kinetic cooperativity observed can be explained in terms of non-equilibrium dynamics between distinct enzyme forms, each capable of performing the catalytic reaction, however with different turnover rates and/or binding affinities. The partitioning of the transient fluxes and the onset of the steady-state are strongly affected by the net-charge of the substrate and its interactions with a pre-formed DNA/enzyme complex. Supported by molecular dynamic simulations, we finally propose a unified linkage scheme that tries to embrace the complexity of the scenarios observed and anticipates a still unknown role of dense DNA surfaces on the transient dynamics of monomeric enzymes.

Host: Professor Kurt V. Gothelf, iNANO & Dept. of Chemistry, Aarhus University