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Glycolysis - Regulation. Lecturer: Rick Kahn RRC G-217 Phone: 7-3561 E-mail: rkahn@emory.edu. Objectives: To begin to think about enzymes as regulated catalysts To understand the different ways enzymes can be regulated

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glycolysis regulation

Glycolysis- Regulation

Lecturer: Rick Kahn

RRC G-217

Phone: 7-3561

E-mail: rkahn@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.

slide3

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

When bookkeeping, double everything

after aldolase

Two steps yield ATP (x2=4), one step reduces

NAD+ to NADH

slide4

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

enzymes are regulated catalysts
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
catalytic sites on enzymes flicker between having a substrate and a product binding sites
Catalytic sites on enzymes “flicker” between having a substrate and a product binding sites

The Substrate

The Enzyme

The Product

simple catalysis
“Simple” catalysis

Enzyme in substrate

binding conformation

Substrate

Chemistry

Happens Here

Product

Enzyme in product

binding conformation

competitive inhibition
Competitive inhibition

Substrate

Inhibitor

Note: Inhibitor can be

an alternate substrate

or simply bind to the

catalytic site

competitive inhibition1
Competitive inhibition

Note: Inhibitor can be

an alternate substrate

or simply bind to the

catalytic site

Substrate

Inhibitor

slide11

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.

---Webster’s 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.

---Stedman’s Medical Dictionary 26th edition

allosteric inhibition binding of a ligand outside the catalytic site can activate or inhibit
Allosteric inhibition:Binding of a ligand outside the catalytic site—can activate OR inhibit

Enzyme with

Allosteric

activating site

Enzyme with

Allosteric

inhibitory site

Enzyme with

Allosteric activation

and inhibitory sites

“Simple” Enzyme

Allosteric

Inhibitor

Allosteric

Activator

Substrate

allosteric activation
Allosteric activation

KD for substrate

0.10 μM

KD for substrate

10 μM

allosteric inhibition
Allosteric Inhibition

In the presence of allosteric

inhibitor, activity is low;

substrate can accumulate

In the absence of the allosteric

inhibitor, activity is high

highly regulated important enzymes can have both allosteric activator and inhibitor sites
Highly regulated (important) enzymes can have both allosteric activator and inhibitor sites

High Activity

Low Activity

Moderate

Activity

a highly specialized rare case activator and inhibitor sites overlap pfk1
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

which steps in a pathway are the ones you would want to regulate
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)
glucose transporters simply facilitate diffusion 0 energy req d
Glucose transporters simply facilitate diffusion (0 energy req’d)
  • Transport would be
  • readily reversible BUT
  • Glucose constantly
  • being metabolized
  • Rapid phosphorylation
  • prevents leakage out
hexokinase and glucokinase each catalyze the same reaction but

Brain

Muscle

Liver

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

slide21

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

phosphofructokinase 2 pfk2 generates fructose 2 6 bisphosphate an allosteric activator of pfk1
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

last step pyruvate kinase is also regulated by glucagon and f1 6bp
Last step (Pyruvate Kinase) is also regulated by glucagon and F1,6BP

When [Glucose]BLOOD

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

slide25

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
summary of glycolysis regulation
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
slide27

Regulation of input and output from glycolytic pathway integrates it with other metabolic pathways

REGULATED

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