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