Desensitization to glucose 6-phosphate of phosphoenolpyruvate carboxylase from maize leaves by pyridoxal 5'-phosphate.

Incubation of the nonphosphorylated form of maize-leaf phospho enol pyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), PEPC, EC 4.1.1.31) with the reagent pyridoxal 5'-phosphate (PLP) resulted in time-dependent, reversible inactivation and desensitization to the activator glucose 6-phosphate (Glc6P) and other related phosphorylated compounds. Both processes are not connected, since (i) when the PLP-modification was carried out in the presence of saturating ligands of the active site, which prevents inactivation, the desensitization to Glc6P is still observed, and (ii) under some experimental conditions the desensitization reaction is 4-times faster than the inactivation. Desensitization to Glc6P is first order with respect to PLP and has a second-order forward rate constant of 4.7 +/- 0.3 s-1 M-1 and a first-order reverse rate constant of 0.0046 +/- 0.0002 s-1. Correlation studies between the remaining Glc6P sensitivity and mol of PLP residues incorporated per mol of enzyme subunit indicate that one lysyl group for enzyme monomer is involved in the sensitivity of the enzyme to Glc6P. The reactivity of this group is increased by polyethylene glycol and glycerol, while the reactivity of the lysyl group of the active site is not affected by these organic cosolutes. In the presence but not in the absence of the organic cosolutes, Glc6P by itself offers significant protection against desensitization, while increases the extent of inactivation. Free PEP or PEP-Mg have opposite effects, protecting the enzyme against inactivation and increasing the degree of desensitization. They also increases the protection against desensitization afforded by Glc6P. Finally, the PEPC inhibitor malate provides some protection against both inactivation and desensitization. Taken together, these results are consistent with PLP-modification of a highly reactive lysyl group at or near the allosteric Glc6P-site.

Flavonoids as inhibitors of NADP-malic enzyme and PEP carboxylase from C4 plants.

The inhibitory effects of flavonoids on the activity of two photosynthetic enzymes such as phosphoenolpyruvate carboxylase (PEPCase) and NADP-dependent malic enzyme (NADP-ME) were evaluated. The glycosylation of hydroxyl groups on the flavonoids resulted in compounds that behaved as gradually weaker inhibitors with increased size of the substituent. Quercetin and baicalein showed a competitive inhibition pattern vs. NADP+ for NADP-ME, and a similar model for both flavonoids vs. phosphoenolpyruvate (PEP) was observed when tested on PEPCase. K(i) for NADP-ME inhibition at pH 7.0 were 0.83 microM and 1.54 microM for quercetin and baicalein, respectively. K(i) for PEPCase inhibition were 0.17 microM and 0.79 microM (quercetin and baicalein, respectively), indicating that these compounds are the most potent inhibitors described for this carboxylase. I50 values for these and other flavonoids were in the micromolar range. A tentative physiological role for the inhibitory effects observed on PEPCase is discussed.

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study.

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.

Stereoselectivity of the interaction of E- and Z-2-phosphoenolbutyrate with maize leaf phosphoenolpyruvate carboxylase.

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 C4-plant P-enolpyruvate carboxylase; the values of Ki were 15-20 microM and 100-110 microM for Z- and E-P-enolbutyrate, respectively. With 0.5 mM Mn2+, the Z isomer was also effective as inhibitor (Ki = 35-40 microM), 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/Km values; however, V and Km for the two isomers were significantly different (i.e. Km = 110 microM for Z-P-enolbutyrate and 220 microM 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.