Stereoselectivity of the interaction of E‐ and Z‐2‐phoshoenolbutyrate with maize leaf phosphoenolpyruvate carboxylase

Abstract

The aim of this work was to investigate the stereoselectivity of maize leaf phosphoenolpyruvate carboxylase with E‐ and Z‐2‐phosphoenolbutyrate as inhibitors and substrates. In addition, a procedure is presented for the separation of the isomers of 2‐phosphoenolbutyrate. 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‐enolbutyrate with high yield. The same system was used to identify Z‐P‐enolbutyrate as the product of the phosphorylation of 2‐oxobutyrate by rabbit muscle pyruvate kinase. In the presence of 5 mM Mg²⁺, both isomers of P‐enolbutyrate inhibited C₄plant P‐enolpyruvate carboxylase; the values of K_i were 15–20 μM and 100–110 μM for Z‐and E‐P‐enolbutyrate, respectively. With 0.5 mM Mn²⁺, 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‐enolbutyrate and 220 μM for E‐P‐enolbutyrate). The results indicate the existence of stereoselectivity for the binding of P‐enolbutyrate to the active site of P‐enolpyruvate carboxylase. However, this fact does not affect the use of the isomers as substrates by the plant carboxylase.