A) Why is it important for cells that proteins are…
A) Why is it important for cells that proteins are degraded instead of remaining indefinitely after being synthesized? (B) Name two ways cells control protein turn over? Hints is in the attachment below CHAPTER 15 Principles of Metabolic Regulation Key topics: Metabolic Pathways •? The biochemical reactions in the living cell — the metabolism — is organized into metabolic pathways •? The pathways have dedicated purposes –? Principles of regulation in biological systems –? Glycolysis vs. gluconeogenesis? –? Chemistry and regulation of glycogen metabolism Metabolic Pathways •? The pathways can be represented as a map Map of Metabolic Pathways •? The biochemical reactions in the living cell — the metabolism — is organized into metabolic pathways •? The pathways have dedicated purposes, some are dedicated –? to extraction of energy –? to storage of fuels –? for synthesis of important building blocks –? to elimination of waste materials •? The pathways can be represented as a map –? Follow the fate of metabolites and building blocks –? Identify enzymes that act on these metabolites –? Identify points and agents of regulation –? Identify sources of metabolic diseases http://www.genome.jp/kegg/ Homeostasis •? Organisms maintain homeostasis by keeping the concentrations of most metabolites at steady state 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 –? increase the capacity of glycolysis during the action –? reduce the capacity of glycolysis after the action –? increases the capacity of gluconeogenesis after successful action 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 •? Regulate fluxes to maintain concentration of metabolite (control enzyme) 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 Feedback Inhibition Flux Control Coefficient for A?D •? Intermediate (branch point) or cofactor (NADPH) can integrate pathways by controlling enzyme early in pathway negative coefficient because branchpoint competes for B relative contribution of each enzymes in a pathway to flux J through pathway, C=0, no impact on J; C=1 total control on pathway Note the Sum = 1 for any system of enzymes analyzed as a group Reactions Far From Equilibrium 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 (segregation, transporters) •? Activity of the catalyst –? Concentration of the enzyme (protein stability, rate of transcription/translation, mRNA/ protein degradation) –? Intrinsic activity of the enzyme (covalent modification, binding of regulatory proteins, expression of isozymes (isoforms) w/ different kinetics and regulation) Rates of a Biochemical Reactions Factors that Determine the Activity of Enzymes •? Rates of a biochemical reactions depend on many factors Michaelis Menten kinetics Rate is ½ Vmax when [S]=Km Enzyme is ½ saturated w/ reactant associate •? Concentration of reactants •? Activity of the catalyst –? Concentration of the enzyme –? Intrinsic activity of the enzyme •? Concentrations of effectors hyperbolic response to concentration –? Allosteric regulators –? Competing substrates –? pH, ionic environment Sensitive to small changes in [substrate] •? Temperature Km These experimental data yield a Km for ATP of 5 mM. The concentration of ATP in animal tissues is ~5 mM. Enzymes from rat liver [E] enzyme C= 0.79 concentration Types of Pathway Management •?metabolic regulation-processes that serve to maintain homeostasis (at molecular level, maintain concentration of a metabolite) example, yeast PFK-1 regulation C= 0.21 C=0 •?metabolic control-process that leads to change in output of pathway in response to a signal (change flux through pathway) example, insulin up regulates glycolytic enzymes transcription Purified enzymes from rat liver added in the amounts shown on the x axis Factors that Determine the Activity of Enzymes transcriptome microarray proteome 2D gel Active Protein Molecules have a Finite Lifespan •? Different proteins in the same tissue have very different half-lives –? less than an hour to about a week for liver enzymes –? The stability correlates with the sequence at N-terminus •? Some proteins are as old as you are –? crystallins in the eye lens Metabolome of E.coli Phosphorylation of Enzymes Affects their Activity •? Protein phosphorylation is catalyzed by protein kinases •? Dephosphorylation can be spontaneous, or catalyzed by protein phosphatases •? Typically, hydroxyl groups of Ser, Thr, or Tyr are phosphorylated •? extracellular signal > •? regulatory cascade > •? Phosphorylated within seconds Some Enzymes in the Pathway Limit the Flux of Metabolites More than Others •? Hexokinase and phosphofructokinase are appropriate targets for regulation of glycolytic flux •? Both far from equilibrium – ?G ‘, so rate sensitive to small changes in [substrate] or [product] WAS thought to be the rate determining step, allosterically regulated by fructose 2,6bisphosphate –? Increased hexokinase activity enables activation of glucose –? Increased phosphofructokinase-1 activity enables catabolism of activated glucose via glycolysis Control of Glycogen Synthesis hexokinase I or II isoform Km 0.1mM so high affinity •? Increased hexokinase activity enables activation of glucose •? glucose 6-phosphate produced in excess E activity turned down •? Glycogen synthase makes glycogen for energy storage •? Insulin signaling pathway –? increases glucose import into muscle –? stimulates the activity of muscle hexokinase –? activates glycogen synthase 4-5mM glucose !metabolite regulation inhibited by glucose 6phosphate Regulation of Hexokinase •? Four isozymes (isoenzymes, isoforms) of hexokinase in humans •? Differ in transcriptional control, distribution in tissues, kinetic and regulatory properties and co-factors required •? Liver- Hexokinase IV, glucokinase, Km 10mM while [Blood glucose] = 4-5mM so low affinity prevents hepatocytes from holding glucose for internal use unless concentrations are high and activity is sensitive to changes at relatively high [glucose] Regulation of Hexokinase IV by Sequestration in Hepatocyte 1) Glut 2 efficient receptor always on membrane so [blood glucose]= [liver cell glucose] Rate of Reaction Depends on the Concentration of Substrates •? The rate is more sensitive to concentration at low concentrations 2) hexokinase IV activity responsive to high [blood glucose] –? Frequency of substrate meeting the enzyme matters –? At 1/2 Vmax enzyme is half saturated •? The rate becomes insensitive at high substrate concentrations –? The enzyme is nearly saturated with substrate 3) hexokinase IV not product inhibited so if excess glucose is around will mop up 4) reversibly bound, negative regulator, held in nucleus . allosteric +effector of binding is fructose 6phosphate while ?effector is glucose 5) as [fructose 6-phosphate] increases hexokinase activity decreases to allow other tissues to get glucose 6) transcriptional regulation hexokinase IV, !ATP, glucogon "low blood glucose, AMP 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 muscle isoform on and saturated liver not retaining glucose liver isoform sigmoidal blood glucose and very responsive to changes in [ ] Elasticity Coefficient Measures the Responsiveness to Substrate Enzyme w/ Typical Michaelis-Menten Kinetics Elasticity coefficient, ? tangent to plot of rate change vs [substrate] At [substrate] below the Km, each increase in [S] produces large increase in the reaction velocity, v. Here enzyme has an ? of about 1.0. At [S] >> Km, increasing [S] has little effect on v; ? here is close to 0.0. Glycolysis vs. Gluconeogenesis Regulation of Phosphofructokinase-1 •? The conversion of fructose-6-phosphate to fructose 1,6bisphosphate is the commitment step in glycolysis •? ATP is a negative effector –? Do not spend glucose in glycolysis if there is plenty of ATP Allosteric regulation, muscle PFK-1 At low [ATP], the K0.5 for fructose 6-phosphate is low, enabling the enzyme to function at a high rate for relatively low [fructose 6phosphate]. 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 When [ATP] is high, K0.5 for fructose 6phosphate is greatly increased, as indicated by the sigmoid relationship between substrate concentration and enzyme activity. Regulation by Fructose 2,6-Bisphosphate •? F26BP activates phosphofructokinase (glycolytic enzyme) •? F26BP inhibits fructose 1,6-bisphosphatase (gluconeogenetic enzyme) F26BP activates phosphofructokinase – Glycolysis F26BP inhibits fructose 1,6-bisphosphatase- Gluconeogenesis 25um [inhibitor] lowers affinity for substrate and enhances AMP inhibition without activator enzyme is not active at physiological [fructose 6phosphate] Regulation by Fructose 2,6-Bisphosphate •? Glycolysis if F26BP is high •? Gluconeogenesis if F26BP is low Regulation of 2,6-Bisphosphate Levels in the Liver •? Insulin/ glucogon hormones •? Xyluloase 5-phosphate from pentose phosphate pathway/ Phosphoprotein phosphatase* ** •? Adenylate cyclase Single protein phosphofructokinase-2 (PFK-2) fructose 2,6-bisphosphatase (FBPase-2) Phosphoprotein phosphatasevariable regulatory subunits for substrate specificity no PO3 Adenylate cyclase Xylulose 5-phosphate Pentose phosphate pathway Molecular Origin of Enzyme Regulation •? Regulation of catalysis typically involves –? Binding of inhibitors, often to the active site –? Binding of regulatory protein subunits phosphoprotein phosphatase 2A (PP2A) determines substrate specificity Microcystin-LR, shown here in red, is a specific inhibitor of PP2A Dephosphorylates bifunctional phosphofructokinase-2 (PFK-2)/ fructose 2,6bisphosphatase (FBPase-2) stimulating glycolysis 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 Glycogen structure and metabolism Glycogen Metabolism •? Glucogen phosphorylase (glucogen break down) •? Phosphoglucomutase glucose-1P # glucose -6P •? Debranching enzyme 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 Epinephrine and Glucagon Stimulate Breakdown Glycogen Synthesis Chapter 15: Summary In this chapter, we learned that: •? 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