3 stages :
-
Stage I - preparatory (Glu 
F-1,6-bisP)
- Energy is invested.
- "Semi-symmetrical" molecule is formed, which can
be cleaved into two three carbon-one phosphate trioses in next
stage.
-
Stage II - oxidative phosphorylation (F-1,6-bisP 2 x 3-PGA)
- Triose phosphates formed by cleavage reaction, then isomerized
to single aldotriose phosphate.
- Aldotriose is oxidized using NAD+ to create a
high energy phosphoric-carboxylic mixed acid anhydride.
- High energy phosphate is transferred from mixed acid anhydride
to ADP to give ATP (2x) and a low energy phosphoglycerate.
-
Stage III - energy generation (2 x 3-PGA 2 x Pyr)
- Primary phosphate ester in 3-PGA is isomerised to a secondary ester (2-PGA).
- Low energy secondary ester is dehydrated (alcohol elimination) to create a high energy enol phosphate.
- High energy phosphate is transferred from enol
to ADP to give ATP (2x) and and an enol.
- The enol tautomerizes spontaneously to Pyruvate, removing the enol product of the previous reaction and driving it forward.
-
Need to regenerate NAD+
Know chemistry/mechanisms for kinases and for isomerase.
Which enzymes are involved in control?
- How are regulatory steps determined experimentally? (Keq. vs. measured Q or
G).
PFK is the Main regulatory enzyme (flux generating step,
first committed step)
- ATP is a strong negative effector of PFK.
- Inhibition by "product" of pathway rather than
enzyme, as expected for flux generating step.
- However, ATP is NOT a physiologically important regulator
of PFK.
- Physiological [ATP] are essentially always too high to allow
enzyme to function, so ATP keeps enzyme "off."
- AMP is a strong positive effector for PFK, overcoming ATP
effects.
- AMP is used as a "proxy" for ATP energy levels
because [ ] changes much greater, thus "amplifying"
changes in [ATP].
- Note requirement for Adenylate Kinase to allow this regulatory
mechanism.
Hexokinase is also a regulatory enzyme
- Regulated by product inhibition by G-6-P.
- Why reaction product inhibition rather than pathway product for HK?
- HK product leads to multiple possible pathways, so no single pathway product should regulate, or catastrophe!
- Note indirect regulation by PFK:
- If PFK shuts down, then G-6-P builds up (if other uses also shut down), tuening off HK.
- If PFK active, then [G-6-P] decreases, allowing HK to resume activity.
What is Energy charge (EC)?
- EC is a (crude, but useful) measure of total adenylate high energy bond availability, and thus cellular energy level
- Why is EC of interest in duscussion PFK (and thus Glycolysis) regulation?
- How is it maintained?
- Be able to calculate E.C.
- What reactions of glycolysis are "irreversible"?
- Note that reactions are irreversible in a physiological sense.
- How can reactions become physiologically irreversible when enzymes catalyze fully reversible reactions, and when their catalytic mechanisms are completely reversible? Review Key
- Which enzymes are involved in control?
- How are regulatory steps determined experimentally? (Keq. vs. measured Q or deltaG).
- PFK is the Main regulatory enzyme (flux generating step,
first committed step)
- Pyruvate Kinase is also regulatory.
- Note there are multiple isozymes with differing regulation.
Only one form of regulation is discussed below.
- PK shows homotropic allosterism towards its substrate,
PEP (sigmoidal kinetics).
- F-1,6-bisP is a positive effector.
- Note that PFK activity also affects PK activity:
- If PFK is active F-1,6-bisP builds up (thermo favorability).
- Feed-forward activator of PK.
- Understand this pathway:
- What kinds of chemical reactions are involved?
- How are they catalyzed?
- Correlation of enzyme names and chemical reactions.
- Which steps consume ATP?
- Which produce ATP?
- Where are NAD+/NADH involved?
- Strategy of reactions in pathway:
- Why split into three carbon pieces?
- Why oxidize?
- Why is PFK main regulatory step?
- Why not first step of pathway?
- Know cofactors used by various enzymes.
- Kinases always (for this course) use Mg2+ (nearly
always true).
- Dehydrogenases in Glycolysis use NAD+/NADH.
- PGA Mutase uses 2,3-bisPGA (BPG or DPG)?
- Why is 2,3-BPG necessary?
- Be able to explain "hi energy" in various compounds
we have discussed in this pathway.
- How can Enolase catalyze a reaction with a free energy of
approximately zero convert a low energy compound into a high
energy compound?
Aldolase mechanism
- Given reactant and products, write and explain mechanism. Review Key
- Key catalytic mechanism is covalent catalysis:
- Draw a "Kinetic Mechanism Diagram" for this reaction.
- Correlate the kinetic and chemical mechanisms.
Glyceraldehyde-3-P DH
- Given the substrates and catalysts, write out the mechanism
for this enzyme. Review Key
- Explain this mechanism in catalytic terms.
- Show reasonable electron movements for bond making/breaking
in this mechanism.
- Draw a "Kinetic Mechanism Diagram" for this reaction.
- Correlate the kinetic and chemical mechanisms.
Review pathway energy generation:
How many ATP equivalents are generated in the conversion of glucose to Lactate?
- Create a chart listing the reactions, the energy products (ATP, NADH, etc.), and the ATP equivalents. Review Key
What are the main entrance/exit points of Glycolysis we
have seen thus far? Review Key
©R A Paselk
Last modified 3 April 2013
