Lecture Connections 15 | Principles of Metabolic Regulation © 2009 Jim-Tong Horng

CHAPTER 15 Principles of Metabolic Regulation –Principles of regulation in biological systems –Glycolysis vs. gluconeogenesis? –Chemistry and regulation of glycogen metabolism Key topics:

Homeostasis Organisms maintain homeostasis by keeping the concentrations of most metabolites at steady state In steady state, the rate of synthesis of a metabolite equals the rate of breakdown of this metabolite

Principles of Regulation The flow of metabolites through the pathways is regulated to maintain homeostasis Sometimes, the levels of required metabolites must be altered very rapidly –Need to increase the capacity of glycolysis during the action –Need to reduce the capacity of glycolysis after the action –Need to increases the capacity of gluconeogenesis after successful action

Feedback Inhibition In many cases, ultimate products of metabolic pathways directly or indirectly inhibit their own biosynthetic pathways –ATP inhibits the commitment step of glycolysis

Reactions Far From Equilibrium are Common Points of Regulation Living systems thrive by keeping some metabolic reactions far from equilibrium while the levels of metabolites are in steady state

Rates of a Biochemical Reactions Rates of a biochemical reactions depend on many factors Concentration of reactants Activity of the catalyst –Concentration of the enzyme –Intrinsic activity of the enzyme Concentrations of effectors –Allosteric regulators –Competing substrates –pH, ionic environment Temperature

Factors that Determine the Activity of Enzymes

Both the amount and catalytic activity of an enzyme can be regulated Chap. 27 Chap. 28

Phosphorylation of Enzymes Affects their Activity Protein phosphorylation is catalyzed by protein kinases Dephosphorylation is spontaneous, or catalyzed by protein phosphatases Typically, hydroxyl groups of Ser, Thr, or Tyr are phosphorylated

Some Enzymes in the Pathway Limit the Flux of Metabolites More than Others Hexokinase and phosphofructokinase are appropriate targets for regulation of glycolytic flux –Increased hexokinase activity enables activation of glucose –Increased phosphofructokinase-1 activity enables catabolism of activated glucose via glycolysis

Control of Glycogen Synthesis Insulin signaling pathway –increases glucose import into muscle –stimulates the activity of muscle hexokinase –activates glycogen synthase Increased hexokinase activity enables activation of glucose Glycogen synthase makes glycogen for energy storage

Regulation of Hexokinase IV by Sequestration

Rate of Reaction Depends on the Concentration of Substrates The rate is more sensitive to concentration at low concentrations –Frequency of substrate meeting the enzyme matters The rate becomes insensitive at high substrate concentrations –The enzyme is nearly saturated with substrate

Isozymes may Show Different Kinetic Properties Isozymes are different enzymes that catalyze the same reaction They typically share similar sequences Their regulation is often different

Glycolysis vs. Gluconeogenesis

Regulation of Phosphofructokinase-1 The conversion of fructose-6-phosphate to fructose 1,6-bisphosphate is the commitment step in glycolysis ATP is a negative effector –Do not spend glucose in glycolysis if there is plenty of ATP

為何 ATP 對 PFK-1 是受質也是 inhibitor? 當 [ATP] 低, 即 [ADP] 高  Low energy charge  glycolysis 當 [ATP] 高, 即 [ADP] 低  High energy charge  glycolysis *[ATP] 低時, ADP 進入 allosteric site 當 activator, ATP 進入 active site 當 substrate *[ATP] 高時, ATP 進入 allosteric site 當 inhibitor + - FIGURE 6–31 Subunit interactions in an allosteric enzyme, and interactions with inhibitors and activators.

Regulation of Phosphofructokinase 1 and Fructose 1,6-Bisphosphatase Go glycolysis if AMP is high and ATP is low Go gluconeogenesis if AMP is low

Regulation by Fructose 2,6- Bisphosphate F26BP activates phosphofructokinase (glycolytic enzyme) F26BP inhibits fructose 1,6-bisphosphatase (gluconeogenetic enzyme)

2 6

Regulation by Fructose 2,6- Bisphosphate Go glycolysis if F26BP is high Go gluconeogenesis if F26BP is low

Regulation of 2,6-Bisphosphate Levels

Regulation of Pyruvate Kinase Signs of abundant energy supply allosterically inhibit all pyruvate kinase isoforms Signs of glucose depletion (glucagon) inactivate liver pyruvate kinase via phosphorylation –Glucose from liver is exported to brain and other vital organs

Two Alternative Fates for Pyruvate Pyruvate can be a source of new glucose –Store energy as glycogen –Generate NADPH via pentose phosphate pathway Pyruvate can be a source of acetyl-CoA –Store energy as body fat –Make ATP via citric acid cycle Acetyl-CoA stimulates glucose synthesis by activating pyruvate carboxylase

Fatty acid oxidation Pyruvate dehydrogenase complex

Glycogen Metabolism

Glycogen synthesis (p565) A polymer of glucose Found in liver and muscle Liver: maintain blood glucose conc. Muscle: glycolytic pathway Why so many branches?

Dealing with Branch Points in Glycogen Glycogen phosphorylase works on non-reducing ends until it reaches four residues from an (  1  6) branch point Debrancing enzyme transfers a block of three residues to the non-reducing end of the chain Debrancing enzyme cleaves the single remaining (  1  6) –linked glucose

Glycogen biosynthesis Base: Glycogenin with glucose in acetal linkage to Tyr-OH Glycogen synthase:  (1 4) linkage of glucose from UDP-glucose UDP-glucose pyrophosphorylase: UTP + G-1- P to UDP-glucose (+PP 2Pi) Branching enzyme: amylo(1,4 1,6) transglycosylase –Chain moved is 6-7 residues long and from 11 residue chain –Branching point:  (1 6) linkage every residues

Fig Fig

Glycogen synthesis Fig

Branch synthesis in glycogen Fig A segment of 6-7 residues is removed from a branch at least 11 residues long and each new branch point must be at least 4 residues (usually 8-12) away from the nearest existing branch point 1. more soluble 2. increases the number of sites accessible to glycogen phosphorylase and glycogen synthase, both of which act only at nonreducing ends.

Epinephrine and Glucagon Stimulate Breakdown of Glycogen

FIGURE Cascade mechanism of epinephrine and glucagon action. By binding to specific surface receptors, either epinephrine acting on a myocyte (left) or glucagon acting on a hepatocyte (right) activates a GTP-binding protein Gsα (see Figure 12-4). Active Gsα triggers a rise in [cAMP], activating PKA. This sets off a cascade of phosphorylations; PKA activates phosphorylase b kinase, which then activates glycogen phosphorylase. Such cascades effect a large amplification of the initial signal; the figures in pink boxes are probably low estimates of the actual increase in number of molecules at each stage of the cascade. The resulting breakdown of glycogen provides glucose, which in the myocyte can supply ATP (via glycolysis) for muscle contraction and in the hepatocyte is released into the blood to counter the low blood glucose.

Chapter 15: Summary living organisms regulate the flux of metabolites via metabolic pathways by –increasing or decreasing enzyme concentrations –activating or inactivating key enzymes in the pathway the activity of key enzymes in glycolysis and gluconeogenesis is tightly regulated via various activating and inhibiting metabolites glycogen synthesis and degradation is regulated by hormones insulin, epinephrine, and glucagon that report on the levels of glucose in the body In this chapter, we learned that: