Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase.

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10-20 microM, assuming a stromal volume of 66 microl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 microM. Malonyl-CoA concentrations were also very low (<0.1-3.0 microM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 microM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA-acyl-carrier protein transacylase reactions at the concentrations of metabolites measured here are < 0.1-4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.