BabcockHall

Twelve pairs of electrons is twenty four electrons. That is enough to reduce six molecules of O2.

I think that if you want to find kcat, you would need both an accurate protein concentration and knowledge that your enzyme was pure.

Are you reading at a single wavelength (280 nm), or are you taking spectra?

Consider a solution of glutamate at its pI (which I seem to recall is about 3.25). At this pH, the ammonium group has a one full (+) charge. The side chain carboxylic acid is mostly neutral, but a small percentage of the time it loses a proton. Therefore it has roughly a charge of 0.1(-). The carboxylic acid that is directly attached to the alpha carbon has a charge of roughly 0.9(-). Thus the positive and negative charges cancel out at this pH. These numbers all depend on one's choice of pKa values.

I would desalt a small portion of the ammonium sulfate precipitate quickly, and run an activity assay on that. Sometimes just centrifuging then removing the supernatant removes most of the ammonium sulfate, and sometimes a Penefsky column works well. I also think starting with a different purification method might be a good idea, as others have said. You might want to pick up Scopes' book and Clarence Suelter's book.

In general the nonesssential amino acids are produced by transaminations of glycolytic or TCA cycle intermediates, or a few extra reactions. The essential amino acids generally have longer pathways. The aspartate pathway and the aromatic amino acid pathway both have many steps.