Turnover number of acetyl-cholinesterase.

The turnover number of acetylcholinesterase is of special interest because the enzyme is thought to be one of the fastest enzymes and because a high speed is a prerequisite for the role of the enzyme in nervous function (1). The earliest value of the turnover number was given by Rothenberg and Nachman sohn (2) in terms of the molecular weight for enzyme obtained from electric eel. The molecular weight was estimated to be 3 x lo6 from the rate of sedimentation in the ultracentrifuge and the preparation with the highest specific activity was assumed to be pure. The turnover number so obtained was 3 x lo7 per minute per molecule of enzyme. Michel and Krop (3) used the irreversible inhibitor, diisopropylfluorophosphate. This compound phosphorylates the active site of acetylcholinesterase (and also that of other enzymes). In that study an enzyme preparation from electric eel was treated with diisopropylfluorophosphate containing radioactive phosphorus. The protein was precipitated with trichloroacetic acid and washed free of unreacted diisopropylfluorophosphate. The radioactivity which remained with the protein gave the number of active sites. h possible error in this method, and one which can be very large, is that diisopropylfluorophosphate, which is a fairly active phosphorylating agent, may phosphorylate other proteins or other groups in addition to the active site. This possibility was discounted when it was found that little radioactivity was carried by the protein when the phosphorylation was carried out in the presence of acetylcholine. The value obtained in that study was 4.9 x lo5 per minute at 0.14 ionic strength, pH 7.4, 38”, and acetylcholine 0.015 M. The value per active complex was calculated to be 7.2 x lo5 per minute. This method was modified by Cohen and Warringa (4) and used with enzyme from human red cells. During phosphorylation with diisopropylfluorophosphate the active site was protected with the poor substrate, butyrylcholine. Excess diisopropylfluorophosphate and butyrylcholine were removed by dialysis and the still active enzyme was finally treated with radioactive diisopropylfluorophosphate. In this way it was hoped that, although other groups might be phosphorylated, only the active site would be phosphorylated with radioactive phosphorus. With the simpler technique, several times as much radioactivity was carried by the protein so that, in this case, the simpler technique was not satisfactory. These studies gave a value of 3 X lo5 per minute per active site. ,4 new method, which is described in this paper, uses dimethylcarbamyl fluoride, which is a very poor substrate and an in-