Allosteric control by calcium and mechanism of desensitization of phosphoenolpyruvate carboxykinase of Escherichia coli.

Phosphoenolpyruvate carboxykinase from Esche- richia coli, strain K12, has been purified by a procedure involving osmotic shock. The molecular weight, using various methods, is estimated to be about 53,000. Different lines of evidence suggest that the enzyme is monomeric. NADH, contrary to earlier reports, is shown not to inhibit the enzyme, but to interfere with the formation of oxalacetate-Fast Violet B complexes in the assay medium. The enzyme is shown to require a divalent metal ion for activity. This requirement is fulfilled by magnesium, manganese, or calcium, although the maximum velocity obtained with each ion varies considerably. When the enzyme is saturated ~ t h magnesium, both Mn2+ and Caz+ further activate the enzymic reaction. The enzyme can be desensitized to calcium activation by treatment with trypsin. This treatment leads to the formation of a larger fragment which has a molecular weight of about 40,000 and is resistant to further pro- teolysis by trypsin. The desensitized enzyme still shows activation by manganese in the presence of magnesium. The maximal velocity obtainable with Mn2+ is also higher with the desensitized enzyme compared to the native form. The binding curve of the calcium analogue, terbium, to the native enzyme is biphasic. It, however, binds very poorly to the desensitized enzyme. It is concluded that calcium has an allosteric site on the enzyme surface which is distinct from the Mn2+ or M 8 + binding sites. (1, P-enolpyruvate’ carboxykinase