1Concepts• Metabolic Regulation– Responsible for correlating the activity of a pathway with the needs of the cell for the end product of that pathway– Kinetic control of enzyme activity accomplishes regulation– Pathways are poised to operate but regulation determines the rate– Regulatory enzyme serves as the rate determining step for a pathway as a whole– Feedback regulation—the concentration of the end products acting on regulatory enzymes helps to correlate the rate to the need for the end products2Terminology• Flux = Rate of flow of intermediates– Flux is determined by “rate determining” steps, which by definition is the slowest step.– Flux must vary to organism’s needsS A B C productVery negative free energy changeVf>> Vr--essentially irreversibleProduct A is removed before it has a chance to be converted back to SS A operates far from the equilibrium3Mechanisms of Regulation1. Allosteric control– Effectors that change the kinetic properties of an enzyme2. Covalent modification (i. e. phosphorylation)2 (or more) forms of an enzyme3. Genetic Controlchange amount of enzyme—long term regulation4Michaelis-Menten Kinetics56Cooperativity+ = positive modulator- = negative modulatorExamples for PFKInhibitors: ATP, citrate, PEPActivators: AMP, ADPEffects of “modulators” on S0.57• Key Enzymes of C.M.P. regulated by adenine nucleotides(experimental observations –1950s 1960s)Significance?Energy Charge HypothesisRegulation of Energy Metabolism by Adenylatesmodifier: + -Phosphorylase AMPPFK AMP ATPADPIsocitrate DH AMP ATPADPThis concept is not covered in the textbook8Energy Charge Hypothesis• Concentration of adenylates in the cell is an indicator of energy status and are modifiers of regulatory enzymes• ATP-regenerating (R) and ATP utilizing (U) pathways to regulate cellular energy status– Cellular energy status“Adenylate Cycle”ATP regenerating pathways (R)ATP Utilizing pathways (U)AMP ADP ATP+ + +PPi Pi H2O121. AMP + ATP ADP + ADP2. PPi + H2O Pi + PiAdenylatekinasepyrophosphatase9Atkinson’s Concepts• ATP synthesis or hydrolysisis involved in most cellular pathways• Pool of Adenylates is constant and limitedPool = [ATP + ADP + AMP]Conversion of ATP, ADP, AMP is very rapidi.e., t½ATP < 1 secAdenylates must be regulating their own synthesis and breakdown by acting as modifiers of regulatory enzyme in ATP regenerating (R) and ATP utilizing (U) reactions/pathways to keep the cycle in a dynamic steady state.10Energy Charge = ECEC = [ATP] + ½ [ADP][AMP] + [ADP] + [ATP]Numerator is charged formsDenominator is totalCellular concentrations:ATP 5 mMADP 1 mMAMP 0.2 mM11Test of Energy Charge Hypothesisisocitrate DHIsocitrate + NAD+αketoglutarate + NADH + H+R-type pathway (regenerates ATP via NADH)AMP, ADP are positive modifiers ATP is a negative modifierMechanism: Adenylates competeat allosteric regulatory site12PhosphofructokinaseFructose-6-P + ATP Fructose-1,6-bisP +ADPR-typeAMP = + modifierMechanism: Adenylates compete at regulatory site13Aspartate kinaseAspartate + ATPAspartate-β-phosphate + ADPU-type pathway (amino acid biosynthesis)ATP = + modifierADP = - modifierMechanism: Adenylates compete at catalytic site14Generalized response of regulatory enzymes• Operation of R and U pathways keeps the charge at a crossover pointR-typeU-type.85 is the crossover pointCrossover point is 0.85.When energy charge moves below 0.85,then R-type pathways predominate.When energy charge moves above 0.85, U type pathways predominate15AMP-dependent Protein Kinase(AMPK) and AEC• This system is activated by cellular stresses that deplete ATP• When AMPK is activated, catabolic pathways turned on• Important in maintaining the “health of a cell”• AMP is the key regulatory molecule16phosphorylasePossible sites of regulation in C.M.P. by A.E.C.Glycogen,starchG-1-PG-6-PF-6-PF-1,6-P2DHAP G-3-P1,3 bis-PGA3-PGA2-PGAPEPPyruvateAcCoAcitrateisocitrateαkgSuccinyl CoASuccinatefumarateOAA12345678910GRRRRRRUUUUR= AEC regulation of an ATP Regenerating PathwayU= AEC regulation of an ATP Utilizing PathwayBranch to biosynthetic pathwayPFKPyrkinasePyr DHCitrate SynthaseIsocitrateDHGlycSynthaseF-1,6-P2 asePyrCarboxylasePEPCarboxykinase17Generalized response of regulatory enzymes• Operation of R and U pathways keeps the charge at the crossover point• Consistency with R and U type pathways• Regulation occurs at branch points• Regulation occurs at both sides of potential “futile” cycles.R-typeU-type18Phosphorylation (Covalent Modificiation)Protein-OH + ATP Protein –OP + ADPTyrosine kinasesSerine, threonine kinasesGlyocgen synthase has many phosphorylation sitesDifferent degree of inactivation, depending on site(s) phosphorylatedProtein kinase A = cAMPdependent protein kinaseDephosphorylationProtein –OP + H2O Protein-OH + Pi Kinases and Phosphatases: Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine,threonine or tyrosine). A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on aprotein. A protein phosphatase catalyzes removal of the phosphate by hydrolysis. Protein kinases and phosphatases are themselves regulated by complex signal cascades. For example, some protein kinases are activated by Ca++-calmodulin. A kinase that has many roles in regulating enzymes of metabolism is Cyclic AMP-Dependent Protein Kinase, also called Protein Kinase A.19cAMP FormationCyclic AMP: cyclic-AMP (cAMP) is well suited to be a transient signal. Synthesis and degradation of cyclic AMP are both spontaneous, but enzymes are required to catalyze these reactions. The enzymes that synthesize and degrade cAMP are regulated. Adenylyl Cyclase (Adenylate Cyclase) catalyzes:ATP à cAMP + PPi The reaction is driven forward by cleavage of PPi, catalyzed by Pyrophosphatase:PPi à 2 Pi Phosphodiesterase catalyzes: cAMP + H2O à AMP(Cleavage by hydrolysis of the bond shown in red is spontaneous, due to stericconstraint in cAMP). Binding of certain hormones (e.g., epinephrine) to the outer surface of a cell activates formation of cAMP within the cell. Cyclic AMP is thus called a second messenger.20Regulation by cAMPHormonal input is into cAMP-dep. Protein kinase because adenylate cyclase is controlled by hormonesATPcAMPhormonesExample: epinephrineR2C22 C + R2-cAMP4(inactive)