Molecular Properties of Phosphoenolpyruvate Carboxylase of Escherichia coli W

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Abstract
The molecular properties of phosphoenolpyruvate carboxylase [EC 4.1.1.31] of Escherichia coli W were studied by physical and chemical methods. The physicochemical properties found are as follows: molecular weight, 361,000; sedimentation coefficient, So20,w=13.3×10−13sec; diffusion coefficient, Do20,w=3.45×10−7cm2.sec−1; apparent partial specific volume, ν=0.736ml.g−1; and molecular ellipticity, [Θ]222=−13,400. The enzyme was found to be composed of 4 identical subunits, each of which contained a single polypeptide chain having a serine residue as the NH2-terminal. Titration experiments with SH-blocking reagents and amino acid analysis of performic acid-oxidized enzyme revealed the presence of 8 moles of SH groups permole of subunit and the absence of disulfide linkages. The enzyme retained its full activity even after modification of 2 SH groups with 5,5′-dithiobis(2-nitrobenzoic acid). Inactivation occurred on titration of 2 additional SH groups with N-ethylmaleimide. The residual 4 SH groups could not be titrated with these reagents without prior denaturation of the enzyme with SDS. The differences in molecular weight and amino acid composition data for the enzymes of three enteric bacteria, Escherichia coli strains W and B, and Salmonella typhimuriuiu strain LT-2 are discussed.

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