Question

What will happen to a reaction that is catalyzed by a particular enzyme if the enzyme has been changed by a mutation?

Answer 1

When the substrate cofactor and enzyme concentration is being increased in an assay, the active site residue mutants reflect measurable activity, although less compared to wild enzyme. The mutant enzymes have demonstrated less affinity for substrate and cofators since the kinetic parameters like Km and Kc values have increased by several fold. That the structural and fucntional impact the higher substrate and cofactor would bring on a mutant that leads inactive enzyme (no activity) to active enzyme (as shows activity). The mutations were like Glu to Ala, Asp, Gln, Asn.

Biological catalysts (that includes proteins, nucleic acids or even lipids) work by accelerating the reaction taking advantage of several physical processes that are contributing to what is commonly described as a transition state stabilization, or equivalently, lowering of the crossing thermodynamic barrier. These processes are:

1. Detailed three dimensional alignments of the incoming reactive atoms of the substrate and the aiding groups of the catalyst (residues of the protein). That applies to the angles, distances, and planes as the orbitals must be disturbed to achieve the final electronic transition leading to the formation or breakage of the bonds.
2. There must be detailed alignments of the charges (or charged groups of the substrate and the catalyst) to facilitate the electron or proton transfer responsible for the realization of the chemical rearrangement. Therefore the active site groups and pH play a crucial role in activity.
3. There must be substantial change in the electrostatic field at the active site to promote the reaction. This is why most of the active sites are located deeply in the protein matrix. By closing active site loops and expelling solvent (water) the dielectric of the medium changes by a factor of 20 making any electrostatic interactions stronger by the same factor thus facilitating the chemical rearrangement which is nothing else but changes in electron distributions.
4. What directly follows from the last two points is that enzymes usually must isolate the active sites from the aqueous environment by closing the loops and expelling mobile waters.
5. The act of catalysis itself is not static and definitely not an equilibrium event (as considered as an individual act), therefore, it is a dynamic nonequilibrium event. The height of the barrier preventing spontaneous chemical transformation varies all the time as well as the velocity of incoming substrate. Therefore, the protein dynamics plays a crucial role in enhancing the fluctuations leading directly to the enhanced rate of crossing the barrier. Almost precisely like a stronger gun promotes shooting through the shielding.

When Glu exchanged for Asp you deviated the distance between the substrate and the crucial oxygen by at least 1 Å. Such changes usually lead to a change of the rate by 100 fold. In changing Glu to Gln or Asp probably changed the polarizability of the oxygen in question (pKa) by at least several pH units which leads to changes on the scale between 100-1000 fold. In Ala mutation probably a spurious water molecule took over the role of a reactive oxygen and lowered the rate by more than 1000 times.

In conclusion there is nothing surprising that mutants have activity and that you were forced to raise significantly the concentration to see it. Concentration is usually a proxy for changing the time or energy state of your system to access the areas that are not available under "normal conditions". This is like rate of nucleosynthesis that is not normally observed (it would constitute a medieval transmutation in alchemy): to have much higher pressure and temperature to get to force the nucleons to coalesce and form new atoms. So we can quite easily transmutate nowdays the elements into each other in nuclear reactors.