Stereoselectivity of the interaction of E‐ and Z ‐2‐phosho enol butyrate with maize leaf phospho enol pyruvate carboxylase

The aim of this work was to investigate the stereoselectivity of maize leaf phospho enol pyruvate carboxylase with E‐ and Z ‐2‐phospho enol butyrate as inhibitors and substrates. In addition, a procedure is presented for the separation of the isomers of 2‐phospho enol butyrate. The method is based on the different interaction of those compounds with a strong anion‐exchange high‐pressure liquid chromatography column using 50 mM potassium phosphate (pH 3) as elution buffer, and allows the obtention of pure E‐ and Z‐P‐enol butyrate with high yield. The same system was used to identify Z‐P‐enol butyrate as the product of the phosphorylation of 2‐oxobutyrate by rabbit muscle pyruvate kinase. In the presence of 5 mM Mg 2+ , both isomers of P‐enol butyrate inhibited C 4 plant P‐enol pyruvate carboxylase; the values of K i were 15–20 μM and 100–110 μM for Z ‐and E‐P‐enol butyrate, respectively. With 0.5 mM Mn 2+ , the Z isomer was also effective as inhibitor ( K i = 35–40 μM), while the E isomer produced activation of the carboxylase probably due to its binding at an allosteric site. Both compounds were substrates of the enzyme with similar V/K m values; however, V and K m for the two isomers were significantly different (i.e. K m = 110 μM for Z‐P‐enol butyrate and 220 μM for E‐P‐enol butyrate). The results indicate the existence of stereoselectivity for the binding of P‐enol butyrate to the active site of P‐enol pyruvate carboxylase. However, this fact does not affect the use of the isomers as substrates by the plant carboxylase.