### Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2007

Lecture Notes: 21 March

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# Thermodynamics in Metabolism, cont.

Last time stopped with relationship of free energy and equilibrium constant. For the special case of equilibrium, the free energy is zero, so G° ' = -RT lnK', G° ' = -5700 ln K (in joules @ 25°C). Thus free energy is related to the equilibrium constant, K. To provide a quantitative feeling for this relationship some values are tabulated below:

 K log K G°' (calories) G°' (joules) 10-3 -3 4,089 17,100 10-1 -1 1,363 5,700 1 0 0 0 103 3 -4,089 -17,100

For non-equilibrium situations we can find the energy available for work using G = G° ' + RT lnQ, where Q is the mass action expression, Q = ([C][D])/([A][B]) for the reaction A + B C + D.

One advantage of using free energy is that it is easier to evaluate the overall equilibrium/energy for a series of sequential reactions (its additive instead of multiplicative): Gtot = Sum[G]. Often use to predict feasibility of pathways, possible energy yields, and to determine when individual reactions are not at equilibrium (important for determining potential control steps etc.).

Note that the overall free energy determines spontaneity of the reaction - the pathway doesn't matter! As noted above thermodynamics is pathway independent. Thus can drive unfavorable reactions by linking with favorable reactions. This can be done:

1. Sequentially: , where the reaction of B to C pulls A to B; or
2. In parallel: , where the two reactions are linked

Example: glucose + phosphate to G-6-P (G= +3300 cal) and ATP + water to ADP + Pi (G= -7600 cal); mix together, no G-6-P (G= -4300 cal). But link with enzyme, Glu + ATP G-6-P + ADP (G= -4300 cal). All of metabolism depends on such coupled reactions. In essence catabolic reactions drive anabolic reactions etc. via direct, and more commonly, indirect, multi-step, coupling.

Metabolism would be extremely complex if coupled processes directly, however. Instead use an intermediate energy carrier: ATP. Thus catabolic processes make ATP which can then be used for anabolic processes, locomotion, pumping ions across cell membranes (major contribution to basal metabolic rate or BMR), etc. Note that ATP is not used to store energy however. (Often compared to electricity's role in our culture).

# Carbohydrate Metabolism

Glycolysis is going to be our first pathway, and it is arguably the most important and universal of the metabolic pathways. Thus we will spend extra time on it, exploring it in some detail from a variety of perspectives. But before we begin glycolysis let's take a brief look at how glucose (and carbohydrate generally) gets to the tissue from food intake.

DIGESTION.

• Initial, limited, breakdown of starch in mouth by salivary amylase
• Low pH of stomach inactivates salivary amylase, denatures many proteins etc.
• In duodenum digestive enzymes are added from the pancreas (pancreatic amylase, lipases, proteases), pH is neutralized, and detergents (bile acids) are added from gall bladder.
• Starches, Proteins, Lipids are all hydrolyzed in duodenum and small intestine where monomers are absorbed across intestinal epithelium. Amino acids and sugars are released into the portal vein to travel to the liver and then the rest of the body.
• First pass through the liver removes sugars and most of the amino acids for processing, storage with the release of glucose and fatty acids over time for use in the peripheral tissues.

# Glycolysis 1

First let's look at Glycolysis to get an overview, then we will look at the reactions and enzymes of this pathway individually. We will then come back and look at the overall regulation and control of this pathway. If we look at the Glycolysis Pathway (overhead), we can break it into three phases:

• Phase 1: phosphorylation and rearrangement of glucose to give a molecule that can be cleaved to two inter-convertible trioses: glucose to F-1,6-bis P (energy invested);
• Phase 2: oxidative phosphorylation of an aldehyde to give a mixed acid anhydride with subsequent transfer of the phosphate to ATP: DHAP & GA-3-P to 3-PGA (energy repaid);
• Phase 3: rearrangement of a phospho-ester to an enol-phosphate with subsequent transfer of phosphate to ATP: 3-PGA to Pyruvate (energy profit). The resulting pyruvate may now go on to the Kreb's TCA Cycle or be converted into lactate. In either case it is critical to regenerate the NAD+ or Glycolysis will come to a halt.

We have looked at the overall pathway of glycolysis (Glycolysis Pathway) and its phases. Now let's note the energy and kinetic relationships of this pathway as shown in Table I. Note the G°' values: some reactions are quite favorable whereas others are unfavorable, but the overall pathway, including triose isomerase, has a net G°' of -44.65 kJ (Glucose to 2 Pyruvates). So Glycolysis is favorable under standard conditions!

 Glycolytic Enzymes . . Brain . Skeletal Muscle . RBC . . G°', kJ K' Q Max Act Q Max Act Q Max Act Hexokinase -21.94 5000 0.04 17 - 1.5 0.00076 0.3 Hex.Isomerase 2.36 0.4 0.22 80 - 176 0.41 5.6 PFK -17.80 1000 0.13 24 - 56 0.044 1.8 Aldolase 23.73 0.0001 0.000002 15 - 78 0.000014 0.7 Triose Isom. 8.29 0.04 - 415 - 2650 0.35 97 GAP DH - - - 105 - 440 - 17.1 PGA K - - - 610 - 169 - 25.6 DH+K -17.22 800 53 - - - 124 - Mutase 4.89 0.15 0.1 122 - 100 0.15 8.6 Enolase -3.23 3.5 3.6 47 - 158 1.7 1.6 Pyr K -23.73 10000 5.4 164 - 387 51 4.6 Lac. DH - - - 100 - 366 - 20.4

Now look at the K' and Q values: remember that K' gives equilibrium values under standard conditions, while Q gives measured values for real tissues. What we want to pay attention to here is differences between these two values (small variations are expected since tissues are not at standard conditions). Here large differences indicate reactions which are not at equilibrium: these reactions must be controlled in some way by the organism! Thus we see large differences for HK, PFK, and PK in brain, and HK and PFK in RBC's. Muscle is like brain (overhead). The G values are plotted below as well for clarity. Finally the max activity column shows us what kind of flux is possible through these enzymes - what does this indicate about these tissues and glycolysis?. (overhead 13.7, MvH)

Figure I. Free Energy changes in rabbit skeletal muscle (Data from Mathews and van Holde, Biochemistry, Benjamin/Cummings (1990))

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