Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate.
4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6%. The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column. At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein). The pH optimum for Vmax is between 7.5 and 7.7. Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme. The enzyme contains covalently bound pyruvate as suggested by the isolation of [3H]lactate and pyruvate from [3H]NaBH4-reduced enzyme and native enzyme, respectively. One mole of [3H]lactate was isolated per 39,000 g of [3H]NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme. Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond. These findings are in accord with similar results obtained with the horse liver enzyme (R. Scandurra, personal communication). The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.
Yang
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Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
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D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008970:Molecular Weight; D009097:Multienzyme Complexes; D010453:Peptide Synthases; D011730:Pyridoxal / Q000494:pharmacology; D011773:Pyruvates / Q000502:physiology*; D019289:Pyruvic Acid
D001894:Borohydrides; C499667:Dfp protein, E coli; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011773:Pyruvates; D011730:Pyridoxal; D019289:Pyruvic Acid; C025364:sodium borohydride; D002262:Carboxy-Lyases; C022373:Phosphopantothenoyl-cysteine decarboxylase; D010453:Peptide Synthases