Gluconeogenesis In animals glucose is required by the brain and is important to the proper functioning of most tissues A fall in plasma glucose can result in unconsciousness and if untreated can be fatal If dietary glucose is insufficient to maintain normal circulating levels of glucose additional glucose must be released from the liver The liver has some glucose stored in the form of glycogen but these stores only last for about 12 hours in the absence of dietary glucose Considerably before the glucose stores have been consumed the organism must begin synthesizing additional glucose from other molecules in a process called gluconeogenesis literally the birth of new glucose The vast majority of gluconeogenesis occurs in the liver with some additional glucose synthesis occurring in the kidney Glucose can be synthesized from pyruvate from nearly all of the standard amino acids or from TCA cycle intermediates Animals cannot convert acetyl CoA to any of these compounds Because the breakdown of fat results nearly exclusively in acetyl CoA fat with minor exceptions cannot be used to synthesize glucose Instead most glucose is synthesized either from lactate produced during anaerobic glycolysis or from amino acids derived from breakdown of proteins Some glucose is synthesized from glycerol a breakdown product of triacylglycerol and phospholipids and some can be synthesized from fatty acids containing an odd number of carbons Cows and other ruminants use a breakdown product of chlorophyll as a gluconeogenic substrate in humans this pathway is present but is far less important In general however gluconeogenesis uses amino acids either derived from dietary protein or from breakdown of proteins stores as the source of the substrate required for the process Enzymes of the gluconeogenic pathway Most of the enzymes used to synthesize glucose are also involved in the glycolytic pathway Some glycolytic reactions however are irreversible under physiological conditions and reversing these steps requires separate enzymes The irreversible steps tend to act as regulatory control points The diagram below summarizes the glycolytic and gluconeogenic pathways The enzymes shown in blue are the regulated glycolytic steps the enzymes in red are regulated gluconeogenic enzymes The enzymes shown in black are common to both pathways The irregular shape at the bottom of the diagram is a mitochondrion Note that the pyruvate carboxylase reaction occurs exclusively in this compartment The phosphoenolpyruvate carboxykinase reaction is shown in the cytoplasm this is true for some species in humans the reaction occurs in both the cytoplasm as shown and in the mitochondria in the latter case the phosphoenolpyruvate is transported out of the mitochondria to allow the remainder of the gluconeogenic reactions to proceed Copyright 2000 2003 Mark Brandt Ph D 28 Pyruvate carboxylase The pyruvate kinase reaction is physiologically irreversible As a result under physiological conditions converting pyruvate to phosphoenolpyruvate requires a short pathway containing two important enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase Pyruvate carboxylase is a mitochondrial enzyme that converts pyruvate to oxaloacetate The pyruvate carboxylase reaction acts as both a mechanism for increasing the amount of TCA cycle intermediates this function will be discussed further in the context of TCA cycle regulation and as the first step of gluconeogenesis Pyruvate carboxylase is a biotin dependent enzyme biotin is covalently bound to the amino group of a pyruvate carboxylase lysine side chain Pyruvate carboxylase catalyzes formation of a covalent between biotin and carbon dioxide in the form of Copyright 2000 2003 Mark Brandt Ph D 29 carbonate in an ATP dependent reaction this carbonate is then transferred to the pyruvate substrate to produce oxaloacetate Because the ATP required for this reaction is derived from catabolism of carbon compounds the pyruvate carboxylase reaction does not involve net carbon fixation Phosphoenolpyruvate carboxykinase Phosphoenolpyruvate is an energetic molecule Its production by phosphoenolpyruvate carboxykinase requires significant energy to form the high energy phosphate bond in the molecule The reaction is driven by GTP hydrolysis in addition the reaction involves the loss of carbon dioxide which acts as an entropic driving force Oxaloacetate cannot leave the mitochondria This is potentially important because the majority of the gluconeogenic enzymes are located in the cytoplasm In order to allow gluconeogenesis to proceed therefore oxaloacetate must be converted to a useful molecule that can be transported out of the mitochondria Two molecules fit this description malate and phosphoenolpyruvate In some animals e g chickens and rabbits the formation of phosphoenolpyruvate by phosphoenolpyruvate carboxykinase occurs in the mitochondria In other animals such as rats and mice the phosphoenolpyruvate carboxykinase is located in the cytoplasm In humans phosphoenolpyruvate carboxykinase is found both in the mitochondria and in the cytoplasm Phosphoenolpyruvate produced in the mitochondria can leave via a specific transporter The alternative to mitochondrial phosphoenolpyruvate synthesis is to produce malate from oxaloacetate because malate can be transported out of the mitochondria In this case cytoplasmic malate dehydrogenase then reforms the oxaloacetate for conversion to phosphoenolpyruvate The use of malate in the cytoplasm has advantages because the conversion of malate to oxaloacetate produces the NADH that will be required for the glyceraldehyde 3 phosphate dehydrogenase step Thus the use of malate transport effectively allows the Copyright 2000 2003 Mark Brandt Ph D 30 transfer of reducing equivalents from the mitochondrion to the cytoplasm In humans the location of phosphoenolpyruvate production is to some extent regulated by availability of NADH in the cytoplasm if the cytoplasmic NADH level is high mitochondrial reducing equivalents are unnecessary for gluconeogenesis and therefore phosphoenolpyruvate is produced in the mitochondria Thought question different gluconeogenic precursors result in phosphoenolpyruvate production in the different locations Which precursor s would favor the use of mitochondrial phosphoenolpyruvate carboxykinase Which precursor s would favor the use of cytoplasmic phosphoenolpyruvate carboxykinase Why The same reversible enzymes that are used in glycolysis