Possible control mechanisms of liver pyruvate carboxylase.

As the first step in the gluconeogenic pathway from pyruvate, the pyruvate carboxylase reaction is an attractive possibility for a control site and studies from the laboratories of Williamson, Park and Soling have indicated that this reaction may be rate-limiting under some circumstances. The control of this enzyme may be complex since the rate of the reaction is affected by a variety of factors including concentrations of substrates (pyruvate, MgATP2-), activators (acetyl-CoA, s-hydroxybutyryl-CoA) and inhibitors (PEP, acetoacetyl-CoA). Our report will describe some of the interactions of these factors at the enzymic level. The studies were conducted mainly with the enzyme isolated from chicken liver but also to some extent with the comparable enzymes from livers of rats, rabbits, calves and turkeys. The Km for pyruvate is about 0.3 mM for this enzyme but the relationship between substrate and initial velocity does not follow Michaelis-Menten kinetics at pyruvate concentrations exceeding 2 mM. At the higher concentrations an apparent substrate activation is observed. It is likely that pyruvate concentrations existing in vivo are in the general range of the Km value and thus that the reaction rate will be affected by changes in pyruvate levels but it should be emphasized that the effects of changes mediated through activators and inhibitors will be superimposed on rate limitations due to pyruvate concentration. The absolute requirement for acetyl-CoA in this reaction has focused attention on this metabolite as a possible control factor. With the avian liver species of the enzyme, the relationship between acetyl-CoA and initial reaction velocity is highly cooperative (Hill constant ≃ 2.9) and the Ka values under optimal conditions are very low (2 μM). Species of the enzyme from mammalian livers (rat, rabbit, calf) differ markedly from the avian species in the quantitative aspects of their interactions with acetyl-CoA. The Hill constants are 2.2-2.3 and the Ka under optimal conditions are an order of magnitude higher (10-20 μM). Under mitochondrial conditions, the Ka values would be expected to increase several-fold and to approach the lower limit of the range of values reported for intramitochondrial acetyl-CoA concentration in rat liver (70-200 μM). A number of inhibitors can increase the apparent Ka for acetyl-CoA. Acetoacetyl-CoA is the only inhibitor found thus far which destroys the cooperative relationship between acetyl-CoA and chicken liver pyruvate carboxylase. At At acetoacetyl-CoA concentrations of 0.5 mM, the Hill constant for acetyl-Coa is reduced to 1 and the apparent Ka for the activator is increased 5-fold. These properties suggest that acetoacetyl-CoA would be an unusually effective control factor for pyruvate carboxylase, particularly since 3-hydroxybutyryl-CoA is an activator of the enzyme. Alterations in the NADH/NAD ratio as reflected in the 3-hydroxybutyryl-CoA/acetoacetyl-CoA ratio could influence the rate of pyruvate carboxylation and gluconecogenesis. At present, the intramitochondrial levels of these acyl-CoA's are not well-established so the validity of this concept cannot be judged. Phosphoenolpyruvate also appeared to be an attractive inhibitor of this enzyme but very recent studies have disclosed that the inhibitor is not phosphoenolpyruvate but an unidentified anionic contaminant which is present in crystalline samples of sodium phosphoenolpyruvate from one commercial source (Calbiochem). The most attractive hypothesis for regulation of pyruvate carboxylation appears to be a multi-layered system of controls with the reaction rate based on available concentrations of pyruvate and/or MgATP2∞ as modified by acetyl-CoA. The effects of the latter would in turn be modulated by the ratio of 3-hydroxybutyryl-CoA/acetyl-CoA. Such a system would provide both excellent sensitivity and flexibility. This work was supported in part by NIH grant R 01 AM 12245 and AEC grant AT-1242.