Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

Kreb's TCA Cycle, cont.

Note that because all catalysts (oxaloacetate, enzymes etc.) must be regenerated in looking at the overall operation of the cycle, only the acetyl group of acetyl-CoA can be oxidized completely.

Integrated Regulation of Aerobic Glucose Metabolism

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 product, as expected for flux generating step.
  - AMP is a strong positive effector for PFK, overcoming ATP effects, used as a "proxy" for ATP energy levels because [ ] changes much greater, thus "amplifying" changes in [ATP].
- Citrate is a negative effector - citrate is a measure of carbon and flux through TCA Cycle, when [ ] is hi then not all carbon is being burned up in cycle, rather it is building up and available for biosynthesis, so slow down production.
- F-2,6-bisP is the most potent positive effector (activator) of PFK in the Liver:
  - F-2,6-bisP is synthesized by PFK-2 which in turn is activated by a hormone dependent phosphorylation by Protein Kinase A,
  - Dephosphorylation activates an independent F-2,6-bisPase active site (on a different domain of the same enzyme);
  - The enzyme activities are also allosterically regulated by various metabolites.
PFK/BisPase "Futile cycle" enhances control in Muscle
- The flux thru glycolysis can change by over 100x
- But [AMP] can only account for about 10x
- PFK rate of 100x and FbisPase 10x
  - Low [AMP] flux = 10-9 = 1 (PFK-BisPase)
  - Hi [AMP] flux = 90-1 = 89
Hexokinase is regulated indirectly by PFK.
- Regulated by product inhibition by G-6-P.
  - G-6-P indicator of glucose use by the various pathways leading from it (Glycolysis, Glycogen synthesis/breakdown, Pentose Phosphate Pathway)
- If PFK shuts down, then G-6-P builds up (if other uses also shut down), turning off HK.
- If PFK active, then [G-6-P] decreases, allowing HK to resume activity.
Pyruvate Kinase is also regulated indirectly by PFK. (Note there are other isozymes with differing regulation.)
- PK shows homotropic allosterism towards its substrate, PEP (sigmoidal kinetics).
- PFK product, F-1,6-bisP, is a positive feed-forward activator of PK.
  - If PFK is active F-1,6-bisP builds up (free energies of glycolysis between F-6P and PEP favor).

TCA Cycle Intermediate Catabolism

Some intermediates, such as citrate, can be partially oxidized, but Kreb's cycle intermediate catabolism requires leaving the cycle at oxaloacetate and then returning as acetyl-CoA. Note that this requires leaving the mitochondria for some reactions, and since the extremely low concentrations of oxaloacetate don't allow its efficient transport across the mitochondrial membrane (the K_m of the carrier is much higher than [oxaloacetate]), malate is the species which actually leaves the mitochondria.

Regulation of the TCA Cycle

In order to understand the regulation of the TCA cycle we need to look at the G values for the various reactions and the kinetic properties of the enzymes. Values for the non-equilibrium reactions are tabulated below:

Data from E. A. Newsholme and A. R. Leach *Biochemistry for the Medical Sciences.* John Wiley & Sons, New York (1983) pp 101 & 110.
Enzyme	Substrate	Substrate Conc. (M)	K_m (M)	G (kJ/mol)	Effectors
Citrate synthase	acetyl-CoA	100-600	5-10	-53.9	Succinyl CoA (-), ATP (-), NADH (-)
Citrate synthase	oxaloacetate	1-10	5-10	-53.9	Succinyl CoA (-), ATP (-), NADH (-)
Isocitrate DH (NAD⁺)	isocitrate	150-700	50-200	-17.5	Ca²⁺ (+), ATP (-), ADP(+), NADH (-)
2-Oxoglutarate DH	2-oxoglutarate	600-5900	60-200	-43.9	Ca²⁺ (+), Succinyl CoA (-), NADH (-)

Note that normally the concentrations of ATP, ADP, NAD⁺, and NADH are relatively constant in the mitosol and are thus unlikely to be very effective as allosteric regulators under most circumstances. On the other hand the availability of NAD⁺ and FAD as substrate will affect the rate not only of the reactions in the table, but also the near-equilibrium dehydrogenases. Note that NAD⁺ availability in turn is determined by the activity of the electron transport system, whose activity is closely coupled to the availability of ADP. Thus high [ATP] will slow the TCA cycle since high [ATP] means low [ADP], which will slow the ETS resulting in low [NAD⁺]!

In muscle, Ca²⁺ does show significant changes in concentration in the mitosol (recall that an increase in [Ca²⁺] concentration initiates muscle concentraction). Succinyl CoA will also show significant concentration changes under differing conditions and can thus also serve as an effective regulator, indicating carbon status in the second half of the cycle.

Regulation and Control: The concentration of citrate also affects PFK activity as a negative effector. You may want to review other aspects of control from last time.

Interconversion of metabolic intermediates: The TCA cycle has a central place in metabolism (even in anaerobic organisms) via its use to interconvert metabolites. We will look further at these interactions as the course proceeds.

Overview of Glucose Metabolism in the Tissues: Diagram in packet.

Metabolism of hexoses other than glucose–Glyc/Gluconeo Overview.

Fructose
Mannose
Galactose on

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. Key

Pathway Diagrams