Regulation of Metabolic Pathways Systems must respond to conditions Homeostasis is not equilibrium Dynamic Steady State –Flux - Rate of metabolic flow of material through pathways Many ways to regulate – for example –At the protein level (e.g. allosteric control) –At the gene level –At transcription or translation There are different time scales for regulation –< sec, seconds, hours, days –Based on situation that requires response

Maintaining ATP concentration is critical –Energy needed to sustain cellular processes –Typical cell [ATP]  5 mM ATP-using enzymes K M range 0.1 – 1 mM Significant [ATP] drop would cause many reactions to decrease Cells are sensitive to ratios ATP/ADP(or AMP) NADH/NAD + NADPH/NADP + ATP + glucose  ADP + glucose 6-phosphate AMP is a very sensitive indicator – small changes make a big difference percentage-wise (normal conc. <0.1 mM)

-Fast response (sec or less) – usually allosteric control (faster response than synthesis or degradation of enzyme) -Covalent modification (also fast) most common: phosphorylation/dephosphorylation -Slower response (sec to hours) –exterior effects such as hormones, growth factors Overall regulatory networks will: 1. maximize efficience of energy source utilization by preventing futile cycles. 2. partition metabolites between alternative pathways (Ex: glycolysis and PPP). 3. use the best energy source for the immediate needs of the cell. 4. shut down biosynthetic pathways when their products accumulate. Vocabulary: Metabolic regulation – maintains homeostasis at the molecular level (e.g. hold concentrations of metabolites constant) Metabolic control – changes flux through a metabolic pathway

Coordinated Regulation of Glycolysis & Gluconeogenesis Futile (substrate) cycles are to be avoided cycles that recycle metabolites at the expense of ATP

Glycolysis Regulation When ATP is needed, glycolysis is activated When ATP levels are sufficient, glycolysis activity decreases Control points 1. Hexokinase 2. PFK-1 3. Pyruvate kinase 1.Hexokinase Hexokinase reaction is metabolically irreversible G6P (product) levels increase when glycolysis is inhibited at sites further along in the pathway Recall there are 4 isozymes G6P inhibits hexokinase isozymes I, II and III Glucokinase (hexokinase IV) forms G6P in the liver (for glycogen synthesis) when glucose is abundant (activity is modulated by fructose phosphates and a regulatory protein)

Isozymes I,II and II have similar K M (important in muscle) –Normally at saturation Hexokinase IV has much higher K M (important in liver) –Important when blood glucose is high

Glucose enters mammalian cells by passive transport down a concentration gradient from blood to cells GLUT is a family of six passive hexose transporters Glucose uptake into skeletal and heart muscle and adipocytes by GLUT 4 is stimulated by insulin Other GLUT transporters mediate glucose transport in and out of cells in the absence of insulin GLUT2 is transporter for hepatocytes Quick equilibrium of [glucose] with blood glucose in both cytosol and nucleus Regulator protein – inside the nucleus –Binds Hexokinase IV and inhibits it –Protein has regulatory site Competition between glucose and fructose 6-phosphate –Glucose stimulates release of hexokinase IV into cytoplasm –Fructose 6-phosphate inhibits this process Hexokinase IV not affected by glucose 6- phosphate as the other isozymes are

Addition of a regulatory protein raises apparent K M for glucose (inhibits hexokinase IV)

Glucose 6-Phosphate Has a Pivotal Metabolic Role in Liver

2. Regulation of Phosphofructokinase-1 Important - this step commits glucose to glycolysis PFK-1 has several regulatory sites ATP is a substrate and an allosteric inhibitor of PFK-1 (note that it’s an end-product of the pathway) AMP allosterically activates PFK-1 by relieving the ATP inhibition (ADP is also an activator in mammalian systems) Changes in AMP and ADP concentrations can control the flux through PFK-1 AMP relieves ATP inhibition of PFK-1

Elevated levels of citrate (indicate ample substrates for citric acid cycle) also inhibit PFK-1 Most important allosteric regulator is fructose 2,6-bisphosphate (later in the chapter) 3. Regulation of Pyruvate Kinase (PK) At least 3 PK isozymes exist in vertebrates Differ in distribution and modulators Inhibited by high ATP, Acetyl-CoA, long-chain fatty acids (energy in good supply) Liver form – low blood sugar  glucagon  increased cAMP  cAMP-dependent protein kinase  PK inactivation (is reversed by protein phosphatase)

Muscle form – epinephrine→increased cAMP → activates glycogen breakdown and glycolysis PK is allosterically activated by Fructose 1,6 BP PK inhibited by accumulation of alanine

Regulation of Gluconeogenesis Fate of pyruvate Go on to citric acid cycle – requires conversion to Acetyl Co-A by the pyruvate dehydrogenase complex Gluconeogenesis – first step is conversion to oxaloacetate by pyruvate carboxylase Acetyl Co-A accumulation inhibits pyruvate dehydrogenase activates pyruvate carboxylase

Coordinated regulation of PFK-1 and FBPase-1 (1) Phosphofructokinase-1 (PFK-1) (glycolysis) (2) Fructose 1,6-bisphosphatase FBPase-1 (gluconeogenesis) Modulating one enzyme in a substrate cycle will alter the flux through the two opposing pathways Two coordinating modulators AMP Fructose 2,6-bisphosphate Inhibiting PFK-1 stimulates gluconeogenesis Inhibiting the phosphatase stimulates glycolysis AMP concentration coordinates regulation stimulates glycolysis Inhibits gluconeogenesis

In the liver, the most important coordinating modulator is fructose 2,6-bisphophate (F2,6BP) It is formed from F6P by the enzyme phosphofructokinase-2 (PFK-2) It is broken down by the same enzyme, but at a different catalytic site in the enzyme – it’s a bifunctional protein -It is called fructose 2,6-bisphosphatase (FBPase-2) for this activity -Balance of PFK-2 to FBPase-2 activity controlled by -Glucagon -Insulin

F2,6BP stimulates glycolysis F2,6BP inhibits gluconeogenesis

Effects of Glucagon and Insulin

The Pasteur Effect Under anaerobic conditions the conversion of glucose to pyruvate is much higher than under aerobic conditions (yeast cells produce more ethanol and muscle cells accumulate lactate) The Pasteur Effect is the slowing of glycolysis in the presence of oxygen More ATP is produced under aerobic conditions than under anaerobic conditions, therefore less glucose is consumed aerobically

Regulation of Glycogen Metabolism Muscle glycogen is fuel for muscle contraction Liver glycogen is mostly converted to glucose for bloodstream transport to other tissues Both mobilization and synthesis of glycogen are regulated by hormones and allosterically Insulin, glucagon and epinephrine regulate mammalian glycogen metabolism (hormones) Ca 2+ and [AMP]/[ATP] (muscle glycogen phosphorylase) [glucose] (liver glycogen phosphorylase) [glucose 6-phosphate] (glycogen synthase) Hormones Insulin is produced by  -cells of the pancreas (high levels are associated with the fed state) -increases glucose transport into muscle, adipose tissue via GLUT 4 transporter -stimulates glycogen synthesis in the liver

Glucagon is Secreted by the  cells of the pancreas in response to low blood glucose (elevated glucagon is associated with the fasted state) -Stimulates glycogen degradation to restore blood glucose to steady-state levels -Only liver cells are rich in glucagon receptors Epinephrine (adrenaline) Released from the adrenal glands in response to sudden energy requirement (“fight or flight”) - Stimulates the breakdown of glycogen to G1P (which is converted to G6P) -Increased G6P levels increase both the rate of glycolysis in muscle and glucose release to the bloodstream from the liver

Reciprocal Regulation of Glycogen Phosphorylase and Glycogen Synthase Glycogen phosphorylase (GP) and glycogen synthase (GS) control glycogen metabolism in liver and muscle cells GP and GS are reciprocally regulated both covalently and allosterically (when one is active the other is inactive) Covalent regulation by phosphorylation (-P) and dephosphorylation (-OH) COVALENT MODIFICATION (Hormonal control) Active form “a” Inactive form “b” Glycogen phosphorylase -P -OH Glycogen synthase -OH -P Allosteric regulation of GP and GS GP a (active form) - inhibited by Glucose GP (muscle)- stimulated by Ca 2+ and high [AMP] GS b (inactive form) - activated by Glucose 6-Phosphate

Hormones initiate enzyme cascades Catalyst activates a catalyst activates a catalyst, etc. When blood glucose is low: epinephrine and glucagon activate protein kinase A Glycogenolysis is increased (more blood glucose) Glycogen synthesis is decreased