Camp. Jeuj. critique

Velayudhan and Kelly
    Bioch 6853
    David Bruce


    The research described in this paper was conducted for the purpose of clarifying the function of certain enzymes of the bacterium Campylobacter jejuni which are involved in gluconeogenic and anaplerotic processes. Campylobacter jejuni is an important cause of gastrointestinal disease in humans worldwide. It also possesses a rather curious set of metabolic pathways as demonstrated by its inability to catabolize glucose, although gluconeogenesis apparently does take place; the organism also contains an enzyme which is homologous to pyruvate kinase, which seems odd because of the irreversible action of this enzyme in glycolytic processes. The authors seek to resolve questions related to enzyme activity and metabolic control within these pathways.
    The authors proceed by developing several mutants, each of which fails to express a specific gene which encodes an enzyme which they are evaluating. Mutants of pyruvate carboxykinase ( pycA and pycB) are generated; each mutant experiences null expression for one or the other portion of the enzyme subunits. Mutants which fail to express pyruvate kinase (pyk) and malic enzyme (mez) are also produced. An attempt was made to create a phosphoenolpyruvate carboxykinase (pckA)  mutant, but this attempt failed as no colonies could be grown. The authors conclude that this is because the organism is unable to survive without the PCK gene product.
    Pyruvate carboxylase is an enzyme which catalyzes the ATP- dependent carboxylation of pyruvate to oxaloacetate. In Campylobacter jejuni it clearly has anaplerotic properties. The pycA mutant’s inability to grow in the absence of malate shows that the net synthesis of oxaloacetate from pyruvate via a malate carbon source is necessary for the organism to achieve a kind of oxaloacetate balance. The growth properties of the pycA and pycB mutants are different because the process of creating the pycB mutant necessarily had a negative effect on the pckA gene which is located a scant 13 base pairs away. The resultant reduction in PCK activity most likely creates a metabolic block with important negative effects on phosphoenolpyruvate (PEP) synthesis. There is apparently no other pathway for synthesizing PEP.
    Malate serves as an energy source in Campylobacter jejuni primarily through its conversion to oxaloacetate in the citric acid cycle, reactions mediated by the enzymes malate dehydrogenase and malate quinone oxidoreductase. In the course of this reaction NADH is produced and ultimately through electron transport leads to ATP synthesis.
    The authors noted that the malic enzyme mutation resulted in little or no effect on the growth of the organism. This is because malic enzyme apparently has a very minor role as an anaplerotic enzyme. It is likely that the introduction of a second mutation, in the malate dehydrogenase gene, would result in a serious growth defect. This mutation would interrupt the citric acid cycle by preventing the synthesis of oxaloacetate from malate; furthermore, in concert with the mez mutation it would leave the organism with no meaningful pathway to the production of oxaloacetate, and thus no pathway to phosphoenolpyruvate, which would be fatal to the organism as demonstrated by the authors’ finding that a pckA mutant failed to colonize. It could be expected that such a mutant would require oxaloacetate in the growth medium, although the accumulation of malate which could be anticipated to take place would probably introduce other variables which would impact the organism’s growth and survival.
    The authors measure kinetic data for these enzymes as well as the impact of other metabolic effectors. This is done because they are interested in learning how these enzymes interact with other substances. Metabolism is not a direct linear process in which one progresses from point A to point B and then to point C. Regulation is multilayered and sometimes complex. Thousands of reactions are taking place simultaneously, some breaking down, some building up (biosynthesis), others creating energy to fuel the whole process, all orchestrated together for the benefit (hopefully) of the organism, which exists holistically on an even more complicated plane.  These reactions and interactions are almost hopelessly intertwined; thus we have anaplerotic enzymes, a focus of this study, which serve to fill gaps left by the “theft” of cycle intermediates by other pathways for biosynthetic purposes, and enzymatic activators and inhibitors (allosteric effectors), which serve to control metabolic reactions by letting an enzyme “know” when it is a good idea to be active or inactive.
    Vmax is the maximum velocity at which a reaction catalyzed by a given enzyme will progress. Km serves as a measurement of the affinity of the enzyme for a substrate. The Hill coefficient is a measurement of cooperative effects induced when the enzyme and substrate interact. The influence of varying concentrations of possible effectors is measured in order to help determine under what conditions the enzyme is inhibited or activated. All of this data is helpful in assigning functions to enzymes and properly evaluating their interplay in the metabolism of Campylobacter jejuni.