Steady-state kinetic analyses indicate that pol β follows an ordered binding of substrates; after binding DNA (Scheme 1, step 1), DNA polymerases preferentially bind a nucleoside triphosphate (dNTP) that preserves Watson Crick hydrogen bonding as dictated by the template (coding) base (step 2).
We employed molecular dynamics simulations to systematically investigate how the individual domain motions are modulated by the binding of substrates.
Additional hydrogen bonding interactions of the protein with GDP may account for the ordered binding of substrates to the enzyme.
The ping pong bi bi kinetic mechanism illustrates alternate binding of substrates and release of products in a bi-substrate reaction with two formed products.
Owing to the network structure of ESM, the mass transfer limitation was suppressed which facilitate the binding of substrates and the release of products.
To help elucidate the phosphorylation mechanism employed by this enzyme, structural changes occurring upon binding of substrates and subsequent catalysis were investigated by protein crystallography.
These conformational changes cycle sequentially through functional states of low, medium, and high nucleotide affinities, corresponding to release of product (ATP), binding of substrates (ADP and Pi), and ATP formation, respectively.
Such mode of interaction seems advantageous for the binding of substrates and efficient catalysis, since in such a dimer the active sites point into the surrounding medium and provide space for the entering substrates.
Analysis of the model indicated amino acid positions in retroviral proteases with the highest influence on ligand activity and revealed general physicochemical properties essential for tight binding of substrates across multiple retroviral proteases.
A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed.
The available crystal structures of the enzyme show non-productive binding of substrates with their ω-end distant from the iron in a hydrophobic pocket at one side of the active site.
