Enzymes catalyze biochemical reactions in highly crowded environments where the amount of macromolecules may occupy up to 40% of the volume. Here we report how cell-like conditions tune catalytic parameters for the monomeric multi-copper oxidase, Saccharomyces cerevisiae Fet3p, in vitro. At low amounts of crowding agent, we detect increases in both of K(M) (weaker substrate binding) and k(cat) (improved catalytic efficiency), whereas at higher crowding levels, both parameters were reduced. Presence of crowding agents does not affect Fet3p structural content but increases thermal resistance. The observations are compatible with ordering of a non-optimal substrate-binding site and restricted internal dynamics as a result of excluded volume effects making the protein less structurally 'strained'.
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