Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate

Abstract

l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that l-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. l-Aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of l-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of l-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. l-Aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, l-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and l-aspartate) are spatially distinct but connected by a network of allosteric interactions.

Global Metabolic Regulation of the Snow Alga Chlamydomonas nivalis in Response to Nitrate or Phosphate Deprivation by a Metabolome Profile Analysis

“…Therefore, we speculated that the aspartic acid increase led to the inhibition of the activities of pyruvate carboxylase, which further demonstrated an inhibitory effect on the TCA cycle. These data are consistent with a previous study that aspartate inhibits pyruvate carboxylase competitively with respect to acetyl-CoA and decreases the levels of the TCA cycle intermediates [ 44 ]. Based on the above results, phosphorus deprivation increases the intermediates of the TCA cycle and reduces the substrates in the Calvin cycle and respiration.…”

The Use of Transposon Insertion Sequencing to Interrogate the Core Functional Genome of the Legume Symbiont Rhizobium leguminosarum

“…Additionally, on TY media mutants in pyruvate dehydrogenase appeared GI, while mutants in pyruvate carboxylase were not. These findings are in agreement with anaplerotic production of oxaloacetate (OAA), via pyc -mediated fixation of CO 2, being important for replenishing OAA pools under minimal growth conditions (Gokarn et al, 2001; Sirithanakorn et al, 2014). …”

Allosteric regulation alters carrier domain translocation in pyruvate carboxylase

“…Unlike acetyl CoA activation, which enhances the coupling efficiency between the two half-reactions in PC, l- aspartate does not alter the enzyme coupling efficiency 8 . In both RePC and AnPC, all three translocation pathways were inhibited by l- aspartate (Tables 4 and 5 ), indicating that the allosteric inhibition by l- aspartate is not mediated through effects on BCCP translocation.…”

“…l -aspartate is a well-characterized allosteric inhibitor of PC. Studies in both RePC and AnPC have suggested that the binding sites for l -aspartate and acetyl CoA are distinct, but that the binding of these two contrasting allosteric effectors is mutually exclusive 8 , 9 .…”

“…As with acetyl CoA, allosteric inhibition by l -aspartate alters the rate of ATP cleavage in the BC domain but, unlike acetyl CoA, it does not alter the coupling efficiency between the individual half-reactions 8 . Thus, while l- aspartate binds competitively with respect to acetyl CoA 8 , 9 , its inhibitory activity does not appear to influence intermediate transfer between active sites.…”