chapter 15 Flashcards
Metabolic Pathways
-The biochemical reactions in the living cell―metabolism―are organized into metabolic pathways
-The pathways have dedicated purposes:
–Extraction of energy
–Storage of fuels
–Synthesis of important building blocks
–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
Metabolic regulation
Maintain homeostasis at the molecular level
Metabolic control-process that leads to a change in the output of a pathway over time in response to a signal or change in circumstances in the cell
Metabolic Pathways
-The pathways are intertwined in a multidimensional network of reactions, e.g. some of the possible fates of glucose 6-phosphate:
-passage into glycolysis for ATP production
-passage to the pentose phosphate pathway for NADPH production
-removal of phosphate to replenish blood glucose
-used to synthesize other sugars such as neuraminic acid for use in protein glycosylation
-partially degraded to make acetyl-CoA for fatty acid and sterol synthesis
-Remember E. coli can use glucose to make the carbon skeleton of every one of its molecules. When glucose is shuttled into one pathway, other pathways may be affected either directly or indirectly
Homeostasis
-Cells and organisms maintain a
-When the steady state is disturbed
-Organisms maintain homeostasis by
-In steady state, the rate of synthesis of a metabolite equals the
-Pathways are at steady state unless
-After perturbation a NEW steady state
-Cells and organisms maintain a dynamic steady state
-When the steady state is disturbed, fluxes through the pathways are changed to return the organism to a new steady state. This is called 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
-Pathways are at steady state unless perturbed
-After perturbation a NEW steady state will be established
Regulatory mechanisms that have been selected that
-maximize fuel utilization by preventing
-partition metabolites appropriately between
-use fuel best suited for the immediate needs of the organism-
-shut down biosynthetic pathways when their
-maximize fuel utilization by preventing simultaneous operation of pathways that work in opposite directions- e.g. glycolysis and gluconeogenesis
-partition metabolites appropriately between alternative pathways- e.g. glycolysis and the pentose phosphate pathway
-use fuel best suited for the immediate needs of the organism- glucose, fatty acids, glycogen, amino acids
-shut down biosynthetic pathways when their products accumulate
Principles of Regulation
-Regulatory mechanisms have evolved to
-The flow of metabolites through the pathways is regulated to
-Sometimes, the levels of required
-Regulatory mechanisms have evolved to ensure that metabolites move through each pathway in the correct direction and at the correct rate to match the organism’s requirements
-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 action
–Need to reduce the capacity of glycolysis after the action
–Need to increase 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
Rates of a Biochemical Reactions
-Rates of a biochemical reactions depend on many factors:
-Concentration of reactants vs. products
-Activity of the catalyst
Concentration of the enzyme
-Rate of translation vs. rate of degradation
Intrinsic activity of the enzyme
-Could depend on substrate, effectors or phosphorylation state
-Concentrations of effectors
–Allosteric regulators
–Competing substrates
–pH, ionic environment
Temperature
Rates of a Biochemical Reactions
-Rates of a biochemical reactions depend on many factors:
-Concentration of reactants vs. products
-Activity of the catalyst
Concentration of the enzyme
-Rate of translation vs. rate of degradation
Intrinsic activity of the enzyme
-Could depend on substrate, effectors or phosphorylation state
-Concentrations of effectors
–Allosteric regulators
–Competing substrates
–pH, ionic environment
Temperature
Proteins have a _____ lifespan
finite
Different proteins in the same tissue have very different half-lives
–Less than an hour to about a week for liver enzymes
–Stability correlates with the sequence at N-terminus
Some proteins are as old as you are
-Crystallins in the eye lens
Phosphorylation of enzymes affects their activity
-Most common type of
-Phosphorylation is catalyzed by
-Dephosphorylation is catalyzed by
-Typically, proteins are phosphorylated on the
-Mechanism is to alter the
-Rapid changes due to
-Most common type of modification
-Phosphorylation is catalyzed by protein kinases
-Dephosphorylation is catalyzed by protein phosphatases, or can be spontaneous
-Typically, proteins are phosphorylated on the hydroxyl groups of Ser, Thr or Tyr
-Mechanism is to alter the active site or to force conformational changes the affect Vmax or Km
-Rapid changes due to allosteric changes that are triggered locally by the concentration of a particular metabolic in the cell
Enzymes are also regulated
by
regulatory proteins
Binding of regulatory protein subunits affects specificity.
Rate of reaction depends on the concentration of substrates
-The rate is more sensitive to concentration at
-The rate becomes insensitive at
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
Km vs. [Metabolite]
-Many enzymes have a Km that is near or greater than the physiological concentration of their substrate
–Especially those utilizing ATP, or NAD(H)
Reactions far from equilibrium are common points of regulation
-Within a metabolic pathway most reactions operate near
Key enzymes operate
-To maintain steady state all enzymes operate at
-Within a metabolic pathway most reactions operate near equilibrium
-Key enzymes operate far from equilibrium
–These are the sites of regulation
–Control flow through the pathway
-To maintain steady state all enzymes operate at the same rate
ATP and AMP are key cellular regulators
-A 10% decrease in [ATP] can greatly affect the activity of ATP utilizing enzymes
-A 10% decrease in [ATP] leads to a dramatic increase in [AMP]
–AMP can be a more potent allosteric regulator
AMP differentially affects pathways
in different tissues via AMPK
Some enzymes in the pathway limit the flux of metabolites more than others
-Enzymes that are
-Not all regulated enzymes have the
-Hexokinase and phosphofructokinase are appropriate targets for
-Enzymes that are far from equilibrium (regulated)
-Not all regulated enzymes have the same affect on the entire pathway
–Some control flux through the pathway
–Others regulate steady state concentrations of metabolites in response to changes in flux
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
Hexokinase affects flux
glycolysis more than phosphofructokinase
Glucose regulation at the organismal level
-Certain tissues do not maintain a glucose
-If blood [glucose] falls from
-Two hormones
-Once glucose arrives at the cell, it must be
-This also helps to illustrate another level of control-
-Certain tissues do not maintain a glucose reserve. E.g. brain needs a supply from blood.
-If blood [glucose] falls from 4 to 5 mM to half level, result in mental confusion and a fivefold reduction results in coma or even death
-Two hormones, insulin and glucagon with opposite effects are used to maintain blood glucose
-Once glucose arrives at the cell, it must be transported across the membrane into the cell. This is a point that can be regulated
-This also helps to illustrate another level of control- that is entry of molecules into cells or cellular compartments can be controlled by the action of molecules that transport them across the membrane
Three reactions of glycolysis are irreversible
- hexokinase
- PFK-1 (phosphofructokinase-1)
- pyruvate kinase
-All three reactions have a large negative ΔG’
-If the forward and reverse of these reactions were catalyzed at the same time, the net effect would be to waste ATP without useful work being done- a large amount of energy would be dissipated as heat and a futile cycle would occur
Hexokinase- a regulatory enzyme
-catalyzes the entry of
-Four isozymes (different proteins that cayalyze the same reaction)
-In muscle the predominant form is
-Blood glucose concentration is about
-Muscle hexokinase I and II are inhibited by
-If cellular concentration of glucose 6-phosphate rises above
-catalyzes the entry of free glucose into glycolysis
-Four isozymes (different proteins that cayalyze the same reaction) I-IV occur each encoded by a different gene
-In muscle the predominant form is hexokinase II which has a high affinity for glucose and is half saturated at 0.1mM.
-Blood glucose concentration is about 4-5 mM so hexokinase II is saturated and is able to work maximally
-Muscle hexokinase I and II are inhibited by their product glucose 6-phosphate.
-If cellular concentration of glucose 6-phosphate rises above normal levels, the isozymes are temporarily inhibited to reestablish the steady state
Glucose commitment to glycolysis is regulated by PFK-1
-The change of fructose 6-phosphate to fructose 1,6-bisphosphate is
-PFK-1 is a complex enzyme with
-ATP is a substrate for
-When [ATP] high= ATP production is
-Conversely when [ADP] or [AMP] increase in the cell, the inhibition of
-The change of fructose 6-phosphate to fructose 1,6-bisphosphate is irreversible and commits glucose to glycolysis
-PFK-1 is a complex enzyme with several regulatory sites where allosteric activators or inhibitors can bind
-ATP is a substrate for PFK-1 and is also an end product of glycolysis and it is used as a regulator for the activity of PFK-1
-When [ATP] high= ATP production is faster than ATP consumption, then ATP binds PFK-1 at an allosteric site and lowers the affinity for fructose 6-phosphate
-Conversely when [ADP] or [AMP] increase in the cell, the inhibition of ATP is released and enzyme activity increases
Other regulators affect PFK-1
-Citrate, a key intermediate in the
-High [citrate] increases the inhibition of
-Citrate serves as an intracellular signal that the cell is meeting its
Fructose 2,6-bisphosphate is also an
-Citrate, a key intermediate in the aerobic oxidation of pyruvate, fatty acids and amino acids also allosterically regulates PFK-1.
-High [citrate] increases the inhibition of ATP, reducing the flow of glucose into glycolysis
-Citrate serves as an intracellular signal that the cell is meeting its current energy needs by oxidation of fats and proteins
-Fructose 2,6-bisphosphate is also an allosteric regulator of PFK-1 in liver
