A STUDY OF THE MOLECULAR SIZES AND STABILITY OF CHOLINE ACETY LTRANSFER ASE

Aggregation of the enzyme acetyl-CoA: choline-O-acetyltransferase (ChAc, EC 2.3.1.6) which appears to be homogeneous has been observed. The molecular weight of the most abundant form of ChAc was estimated by gel filtration and sucrose gradient centrifugation to be in the range 58,000-62,000. The most frequently encountered aggregates were much larger and eluted in the void volume from Sephadcx® G-100 and G-150 indicating molecular weights in excess of 400,000. In fact, they were subsequently found to be 1.2 × 10⁶ and 1.9 × 10⁶ by sucrose gradient centrifugation. The percentage of activity associated with high molecular weight ChAc increased with purification, but these aggregates disappeared after storage for 2-3 weeks at ?20°C. The loss occurred independently of any fall in enzymic activity in the preparations examined.     

THE SYNTHESIS OF ACETYLCHOLINE FROM ACETYL-CoA, ACETYL-DEPHOSPHO-CoA AND ACETYLPANTETHEINE PHOSPHATE BY CHOLINE ACETYLTRANSFERASE

Abstract— The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetyl-CoA, acetyl-dephospho-CoA and acetylpantetheine phosphate. At pH 7.5 K_m values of 25.7 μ m for acetyl-CoA, 54.8 μ m for acetyl-dephospho-CoA and 382 μ m for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3-phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase.
Inhibitor constants ( K_i ) for CoA, dephospho-CoA and pantetheine phosphate were also measured and when considered with the K_m values obtained for the acetyl derivatives it is concluded that acetyl-dephospho-CoA could be a successful acetyl donor in the synthesis of ACh.
Acetyl-dephospho-CoA was found to be less satisfactory as a substrate for citrate synthase.