The Isolation and General Properties of Escherichia coli Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

Frank E Ruch,P Roy Vagelos

doi:10.1016/s0021-9258(19)43197-9

Abstract

Abstract Malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli was purified 4800-fold by procedures which included chromatography on DEAE-cellulose, Sephadex G-100, Sephadex G-75, DEAE-Sephadex, and preparative polyacrylamide gel electrophoresis. The purified enzyme was shown to be homogeneous by electrophoresis on polyacrylamide gels, by sedimentation equilibrium centrifugation, and by amino acid analysis. A molecular weight of 36,660, obtained with carboxymethylated enzyme by sedimentation equilibrium measurements, agreed with determinations on the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (36,500) and by Sephadex G-100 column chromatography (37,000). An s20,w = 2.31 S was determined for the enzyme by sedimentation velocity measurements. Amino acid analysis and determination of the isoelectric point (pH 4.65) both showed the enzyme to be acidic. The purified enzyme was inhibited by N-ethylmaleimide and to a lesser extent by iodoacetamide, but inhibition by both reagents was significantly increased at pH values above 7.3. Preincubation of the enzyme with malonyl-CoA protected against inactivation by both sulfhydryl reagents. Reduction of aged enzyme preparations by incubation with dithiothreitol was shown to stimulate enzyme activity approximately 5-fold. These experiments suggest that a reduced sulfhydryl group(s) on the enzyme is required for maximal catalytic activity.

Highlights

Preincubation of the enzyme with malonyl-CoA protected against inactivation by both sulfhydryl reagents
Reduction of aged enzyme preparations by incubation with dithiothreitol was shown to stimulate enzyme activity approximately S-fold. These experiments suggest that a reduced sulfhydryl group(s) on the enzyme is required for maximal catalytic activity
Malonyl Transacylase Assays-Two assay procedures were used for the measurement of malonyl transacylase activity

Summary

Methods

Malonyl Transacylase Assays-Two assay procedures were used for the measurement of malonyl transacylase activity. The formation of malonylACP by malonyl transacylase was coupled to the condensation of malonyl-ACP and acetyl-ACP by P-ketoacyl-ACP synthetase and the reduction of acetoacetyl-ACP by &hydroxyacyl-CoA dehydrogenase in the presence of DPNH. The routine assay mixture contained 10 pmoles of potassium phosphate, pH 7.0, 2.5 pmoles of dithiothreitol, 0.1 pmole of EDTA (pH 7.0), 30 nmoles of DPNH, 0.4 ~g of fl-hydroxyacyl-CoA dehydrogenase (195 pmoles per min per mg of protein at 37”), 10 nmoles of reduced ACP, 10 nmoles of acetyl-ACP, 15 nmoles of malonyl-. CoA, 5 pg of P-ketoacyl-ACP synthetase (3 pmoles per min per mg of protein at 25”), and 0.08 to 0.8 milliunits of malonyl transacylase in a total volume of 0.1 ml. A unit of enzyme activity is defined as an amount of enzyme catalyzing the formation of 1.0 pmole of DPN per min under the assay conditions.

Results

Discussion

Conclusion