Glycolysis - Regulation

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Glycolysis - Regulation

• Lecturer Rick Kahn
• RRC G-217
• Phone 7-3561
• E-mail rkahn_at_emory.edu

Objectives To begin to think about enzymes as
regulated catalysts To understand the different
ways enzymes can be regulated To learn the key,
regulated steps in glycolysis, the mediators of
regulation, and how it is connected to other
pathways So today we will talk first about
general features of metabolic/enzyme
regulation and then the specifics as they relate
to glycolysis.
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Glycolysis is just one of many pathways all going
on in a cell
YOU ARE HERE
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The Glycolytic Pathway
10 enzymatic steps get you from glucose to
pyruvate Phosphorylation of glucose and fructose
6-phosphate require input of 2 ATPs Cleavage of
fructose 1,6 bisphosphate (hexose) yields two
trioses, only one of which is further metabolized
in glycolysis but the two are interconvertible Wh
en bookkeeping, double everything after
aldolase Two steps yield ATP (x24), one step
reduces NAD to NADH
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Hypothetical Metabolic Pathway
In enzyme-limited steps the substrate accumulates
In substrate-limited reactions the substrate is
quickly metabolized so does not accumulate
Regulation can only occur at sites that are
enzyme-limited
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Enzymes are REGULATED catalysts

• Regulation can occur as a result of
• limiting substrate availability - OFTEN
• limiting enzyme concentration Genetic disease?
• limiting turnover rate different isoforms?
• covalent modification of the enzyme (e.g.
  phosphorylation)often related to endocrine
  control
• through interaction(s) with modulators that bind
  to the substrate binding site (competitive
  inhibition)
• to the product binding site (product inhibition)
• or another, termed allosteric, site, that can be
  stimulatory or inhibitory Key concept

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Catalytic sites on enzymes flicker between
having a substrate and a product binding sites
The Substrate
The Enzyme
The Product
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Simple catalysis
Enzyme in substrate binding conformation
Substrate
Chemistry Happens Here
Product
Enzyme in product binding conformation
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Competitive inhibition
Substrate
Inhibitor
Note Inhibitor can be an alternate substrate or
simply bind to the catalytic site
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Competitive inhibition
Note Inhibitor can be an alternate substrate or
simply bind to the catalytic site
Substrate
Inhibitor
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Product InhibitionExcess product ties up the
enzyme in one state and thus slows catalysis
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Allosterism
Alteration of the activity of a protein (as an
enzyme) by combination with another substance at
a point other than the chemically active
site. ---Websters 9th Collegiate Dictionary The
influence to an enzyme activity brought about by
the change in conformation of the protein in
response to the binding of a substance or other
effector at a site other than the active
site. ---Stedmans Medical Dictionary 26th edition
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Allosteric inhibitionBinding of a ligand
outside the catalytic sitecan activate OR inhibit
Enzyme with Allosteric activating site
Enzyme with Allosteric activation and
inhibitory sites
Enzyme with Allosteric inhibitory site
Simple Enzyme
Allosteric Activator
Allosteric Inhibitor
Substrate
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Allosteric activation
KD for substrate 0.10 µM
KD for substrate 10 µM
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Allosteric Inhibition
In the absence of the allosteric inhibitor,
activity is high
In the presence of allosteric inhibitor, activity
is low substrate can accumulate
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Highly regulated (important) enzymes can have
both allosteric activator and inhibitor sites
High Activity
Low Activity
Moderate Activity
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A highly specialized (rare) case activator and
inhibitor sites overlap (PFK1)
Activator e.g., ADP
High Activity
Gamma phosphate of ATP
PFK1
Low Activity
Inhibitor ATP
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Which steps in a pathway are the ones you would
want to regulate?

• Entry to the pathway (the committed step)--PFK1
• Exit from a pathway--Pyruvate Kinase
• Entry/exit points within one pathway that go to
  or come from another pathway--Hexokinase (kind of)

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Glucose transporters simply facilitate diffusion
(0 energy reqd)

• Transport would be
• readily reversible BUT
• Glucose constantly
• being metabolized
• Rapid phosphorylation
• prevents leakage out

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Hexokinase and Glucokinase each catalyze the same
reaction but
Hexokinase is subject to product inhibition but
glucokinase is not When glycolysis (PFK1) is
inhibited, the pathway gets constipated and backs
up, resulting in shut down of hexokinase
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The primary site of regulation of the glycolytic
pathway is PFK1
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PFK 1 can sense the energy charge of the cell
because both ATP (inhibitory) and ADP
(stimulatory) are allosteric modulators
Glucose 6-phosphate
Regulators of PFK1 Adenine nucleotides (ATP,
ADP) Citrate F2,6 bisphosphate pH
Fructose 6-phosphate
Phosphofructokinase 1 (PFK1)
_

Fructose 1,6-bisphosphate
_
Glycolysis
ATP
ADP
Work
Pyruvate
Citrate
TCA cycle
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Phosphofructokinase 2 (PFK2) generates fructose
2,6-bisphosphate, an allosteric activator of PFK1
Glucose 6-phosphate
Fructose 6-phosphate
Fructose 2,6-bisphosphatase
PFK1
PFK2

Fructose 1,6-bisphosphate
Fructose 2,6-bisphosphate
Fructose 2,6-bisphosphate is a dead end and can
only be cleaved by phosphatase back to fructose
6-phosphate Why, why, why? Why is there PFK2 and
fructose 2,6 bisphosphate? Answer PFK2 is the
supercharger of glycolysis.
Glycolysis
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PFK2 allows for endocrine (insulin and glucagon)
regulation of glycolysis
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Last step (Pyruvate Kinase) is also regulated by
glucagon and F1,6BP
When GlucoseBLOOD is low, glucagon is up, PKA
is active, liver PK gets phosphorylated, and is
turned off this is part of the switch to
gluconeogenesis Fructose 1,6 bisphosphate is
an allosteric activator of PK and ATP is an
allosteric inhibitor
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Summary of Endocrine Regulation (of
carbohydrate/glucose metabolism)

• Insulin and Glucagon are the main endocrine
  factors (each is a protein hormone) that modulate
  blood glucose levels and they act
  antagonistically
• The Pancreas secretes Insulin (? cells) and
  Glucagon (? cells)
• Insulin
• In liver it ? production of glucose by ?
  gluconeogenesis and glycogenolysis
• In muscle and liver it ? glycogenesis
• In muscle and fat cells it ? uptake of glucose
• Glucagon
• In liver it ? glycogenolysis and gluconeogenesis

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Summary of Glycolysis Regulation

• Three enzymes involved are PFK1, PK, and
  hexokinase (and PFK2)
• The changes all make sense.
• When there is plenty of ATP in the cell, slow it
  down to allow storage of glycogen in the liver
  thus hexokinase off, PFK off (ATP and citrate
  allosteric inhibition, little ADP to activate).
• When blood glucose is high, insulin is up, PFK1
  is on, PFK2 is on (further activating PFK1), PK
  is on
• When blood glucose is low, most cells go into
  energy sparing mode to conserve for
  brain/muscle glucagon is up, PFK1 is off, PFK2
  is off, PK is off

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Regulation of input and output from glycolytic
pathway integrates it with other metabolic
pathways
Note metabolism of fats feeds into TCA cycle
downstream of pyruvate this is why it is
important to have citrate as an allosteric
inhibitor of glycolysis