Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent) : transition metal ion requirement for activity and sulfhydryl group reactivity

Abstract

We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phospho enolpyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg 2+ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co 2+ > Mn 2+ > Cd u2+ > Ni 2+ ⪢ Fe 2+ > VO 2+, while Zn 2+ and Ca 2+ had no activating effects. When we investigated the effect of varying the type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO 2 substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn 2+ activated enzyme the inhibition was purely non-competitive ( K ii = K is) towards all substrates, while for the Co 2+-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5′-dithiobis(2-nitro-benzoate) (DTNB) lead to an almost complete inhibition of Mn 2+-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO 3 − had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co 2+-activated enzyme than for its Mn 2+-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn 2+ and ADP afforded almost complete protection from inactivation; in the presence of Co 2+ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.