ISOLATION AND CHARACTERIZATION OF A NATURALLY OCCURRING COFACTOR OF CARBAMYL PHOSPHATE BIOSYNTHESIS

Abstract

Optimal conditions for the biosynthesis of n-citrulline by preparations obtained from mammalian liver require the addition of ATP,' n-ornithine, ammonia, bicarbonate, magnesium ion, and an appropriate derivative of n-glutamic acid (1, 2).Of the many synthetic compounds tested for catalytic activity, N-acetyl-, N-chloroacetyl-, N-carbamyl-, N-propionyl-, and N-formyl-n-glutamic acids have been found to be active (1,2).Acetyl and carbamyl n-glutamates are catalytically active in the biosynthesis of carbamyl phosphate, an intermediate precursor of the carbamyl group of L-citrulline (l-3).It was considered important to determine which, if any, of the synthetic glutamyl derivatives is the naturally occurring cofactor for carbamyl phosphate synthesis.Grisolia and Cohen (4) have previously reported that citrulline synthesis of rat liver preparations occurred only if the enzyme preparation is fortified with a glutamyl derivative.Jones et cd.( 5), using comparable beef liver preparations, confirmed this finding.Lowenstein and Cohen (6) have reported that the requirement with crude enzyme preparations is not absolute.It is possible to demonstrate a small, but significant, synthesis of citrulline by rat liver preparations in the absence of an added glutamyl derivative (Fig. 1).Although the amount of synthesis is only 1.5 to 2.0 per cent of that obtained if the system is supplemented with acetyl glutamate (Fig. 2), it was suspected that this amount of synthesis must be due to the presence of a cofactor in the rat acetone powder extracts.