Carbohydrate anabolism We have covered some aspects of carbohydrate catabolism: glycolysis, PPP, citric acid cycle, etc. and now we turn to carbohydrate anabolic pathways that utilize ATP and reducing power for biosynthesis (ATP used to make favorable reactions) –Anabolic pathways are generally reductive rather than oxidative We will use this tact in the future to cover the metabolism of amino acids, lipids, etc.

Which way am I going? It’s easiest to consider metabolic pathways as simple linear processes where A leads to B, B to C, etc. BUT, anabolic and catabolic pathways proceed simultaneously (albeit at different rates) producing a dynamic steady state

Levels of organization Although they may share many reactions, biomolecules are synthesized and degraded via different pathways. Each catabolic and anabolic pathways has at least one unique enzymatic reaction that is essentially irreversible If not for this, flux through metabolic pathways would solely be due to mass action

Unique reactions are points of control Like other pathways, a biosynthetic pathway is usually regulated at an early step that commits intermediates to that pathway Opposing (catabolic and anabolic) pathways are regulated in coordinated reciprocal manners

Citric acid cycle and glyoxylate cycle Isocitrate conversion is the point of control between these two pathways Accumulation of citric acid cycle intermediates activate isocitrate dehydrogenase Accumulation of citric acid cycle intermediates inhibits isocitrate lyase

Carbohydrate biosynthesis

Gluconeogenesis A seemingly universal pathway “reverse” glycolysis; Pyruvate  glucose Seven of the ten reactions of gluconeogenesis are the reverse of glycolytic pathways Three glycolytic steps are essentially irreversible under cellular conditions –Hexokinase, PFK-1, pyruvate kinase

These three reactions are “bypassed” Pyruvate  PEP Fructose 1,6 bisphosphate  Fructose 6- phosphate Glucose 6-phosphate  glucose

First “by-pass” involves two steps Instead of pyruvate kinase, phosphorylation of pyruvate is accomplished by through intermediate stages involving oxaloacetate and malate Pyruvate is transported from cytosol to mitochondria (or generated from alanine within mitochondria via transamination)

Pyruvate carboxylase is the first regulated step in gluconeogenesis This biotin-containing enzyme was introduced via anaplerotic reactions Pyruvate carboxylase requires acetyl-CoA as a positive effector Oxaloacetate is formed through this reaction, which is subsequently reduced to malate via malate dehydrogenase and NADH

Malate serves as a shuttle for oxaloacetate The resulting malate is transported to the cytosol via the malate –  -KG transporter (from aspartate-malate shuttle) In the cytosol, malate is re-oxidized to OAA by cytosolic MDH OAA is converted to PEP by phosphoenolpyruvate carboxykinase

From pyruvate to PEP

Note the investment in activation of intermediates through this reaction One ATP and one GTP used, contrasting the single ATP used to make PEP in glycolysis The CO 2 added in the first reaction is released in the second

Why go thru the mitochondria? The [NADH]/[NAD] ratio in the cytosol is ~10 5 times lower than in mitochondria, gluconeogenesis relies on NADH Transport of malate (reduced form of OAA) facilitates transport of reducing power from mitochondria to cytosol (subsequent generation of NADH by MDH) to aid in gluconeogenesis

A second PEP biosynthetic pathway Lactate, instead of pyruvate, serves as a starting substrate in some situations (anaerobic muscle or erythrocytes) Conversion of lactate to pyruvate generates NADH obviating the need to export reducing power from mitochondria As a result, the PEP is generated within the mitochondria

The second and third “by-pass” are similar Fructose 1,6-bisphosphate is converted to fructose 6-P by fructose 1,6-bisphosphatase Glucose –6-phosphate is converted to glucose by glucose 6-phosphatase These reactions do NOT result in ATP formation, instead the irreversible hydrolysis forming inorganic phosphate

The cost of gluconeogenesis

Many molecules can feed into gluconeogenesis This is of importance when we get to amino acid biosynthesis

Reciprocity of glycolysis and gluconeogenesis Simultaneous operation of both glycolytic and gluconeogenic reactions would be wasteful if both reactions proceed at high rates in cells (The “simultaneous” operation of anabolic and catabolic pathways is a regulated process) Futile cycles can be engaged for physiological purposes such as heat energy

Reciprocal regulation The first control point for regulating flux between these pathways is pyruvate Pyruvate can be converted to acetyl-CoA (pyruvate dehydrogenase) or to OAA (pyruvate carboxylase) Acetyl-CoA is a positive allosteric effector of pyruvate carboxylase and a negative modulator of pyruvate dehydrogenase

Effects of acetyl-CoA

A regulatory example When cells have enough energy, oxidative phosphorylation slows, NADH accumulates, inhibits the citric acid cycle and acetyl-CoA accumulates. This directs pyruvate to gluconeogenesis

A second control point Fructose 1,6-bisphosphatase is strongly inhibited by AMP, while PFK-1 is activated by AMP and ADP, but inhibited by citrate and ATP Again, these opposing steps are regulated in coordinated and reciprocal fashion Also, hormonal regulation in the liver

Hormonal regulation is mediated by fructose 2,6 bisphosphate fructose 2,6 bisphosphate is an allosteric effort for PFK-1 and fructose 1,6- bisphosphatase fructose 2,6 bisphosphate binds and increases PFK-1 affinity for fructose 6- phosphate, and reduces it’s affinity for ATP and citrate – stimulating glycolysis fructose 2,6 bisphosphate inhibits fructose 1,6-bisphosphatase

Fructose 2,6-bisphosphate regulation

Fructose 2,6-bisphosphate formation regulation Fructose 2,6 bisphosphate is generated by PFK-2 and broken down by fructose 2,6 bisphosphatase (single polypeptide) – note this compound is not a metabolic intermediate, but a regulatory compound

Glucagon lowers the cellular level of fructose 2,6-bisphosphate Inhibits glycolysis, but stimulates gluconeogenesis Process occurs via a signal transduction pathway, which results in alteration of PFK-2/FBPase-2 polypeptide