1.5.4. Coupled Reaction

Unit 1 1. The cytosolic NADH reduces oxaloacetate to malate. 2. The malate is transported into the matrix through a malate-α-ketoglutarate carrier (antiport).

3. The transported malate reduces the NAD+ in the matrix to NADH, and becomes oxaloacetate. 4. The oxaloacetate receives an amino group from a Glu, and becomes Asp. The deaminated Glu becomes a α-ketoglutarate. 5. Asp in the matrix is transported into the cytosol through a glutamate-aspartate carrier (antiport).

Figure 1.5.13. Malate-aspartate shuttle. This shuttle for transporting reducing equivalents f rom cytosolic NADH into the mitochondrial matrix is used in liver, kidney, and heart. 1 NADH in the cytosol (intermembrane space) passes two reducing equivalents to oxaloacetate, producing malate. 2 Malate crosses the inner membrane via the malate–ketoglutarate transporter. 3 In the matrix, malate passes two reducing equivalents to NAD, and the resulting NADH is oxidized by the respiratory chain. The oxaloacetate formed f rom malate cannot pass directly into the cytosol. 4 It is f irst transaminated to aspartate, which 5 can leave via the glutamate-aspartate transporter. 6 Oxaloacetate is regenerated in the cytosol, completing the cycle.

6. The transported Asp donates its amino group to α-ketoglutarate, and Asp becomes oxaloacetate, and αketoglutarate becomes Glu.

1.5.4. Coupled Reaction

 A spontaneous reaction may drive a non-spontaneous reaction.  Free energy changes of coupled reactions are additive. Examples of different types of coupling: A. Some enzyme-catalyzed reactions are interpretable as two coupled half-reactions, one spontaneous and the other non-spontaneous. At the enzyme active site, the coupled reaction is kinetically facilitated,

Unit 1 while the individual half-reactions are prevented. The free energy changes of the half-reactions may be summed, to yield the free energy of the coupled reaction. For example, in the reaction catalyzed by the Glycolysis enzyme Hexokinase, the two half-reactions are:  ATP + H2O  ADP + Pi.................. DGo' = -31 kJoules/mol  Pi + glucose  glucose-6-P + H2O ... DGo' = +14 kJoules/mol

Coupled reaction:

ATP + glucose  ADP + glucose-6-P.. DGo' = -17 kJoules/mol

 The structure of the enzyme active site, from which water is excluded, prevents the individual hydrolytic reactions, while favoring the coupled reaction. B. Two separate enzyme-catalyzed reactions occurring in the same cellular compartment, one spontaneous and the other non-spontaneous may be coupled by a common intermediate (reactant or product). A hypothetical, but typical, example involving pyrophosphate:  enzyme 1: A + ATP  B + AMP + PPi ....DGo' = +15 kJ/mol  enzyme 2: PPi + H2O  2 Pi ....................DGo' = –33 kJ/mol Overall: A + ATP + H2O  B + AMP + 2Pi ... DGo' = –18 kJ/mol

 Pyrophosphate (PPi) is often the product of a reaction that needs a driving force. Its spontaneous hydrolysis, catalyzed by Pyrophosphatase enzyme, drives the reaction for which PPi is a product.  For an example of such a reaction, see the discussion of cAMP formation below. o 3',5'-Cyclic AMP (abbreviated cAMP),is used by cells as a transient signal. o Adenylate Cyclase (Adenylyl Cyclase) catalyzes cAMP synthesis: o ATP  cAMP + PPi. o The reaction is highly spontaneous due to the production of PPi, which spontaneously hydrolyzes. C. Ion transport may be coupled to a chemical reaction, e.g., hydrolysis or synthesis of ATP.

 It should be recalled that the ATP hydrolysis/synthesis reaction is

ATP + H2O  ADP + Pi.  The free energy change (electrochemical potential difference) associated with transport of an ion S across a membrane from side 1 to side 2 is represented below.

R = gas constant, T = temperature, Z = charge on the ion, F = Faraday constant, and DY = voltage across the membrane.

 Since free energy changes are additive, the spontaneous direction for the coupled reaction will depend on the relative magnitudes of:DG for the ion flux (DG varies with the ion gradient and voltage.)DG for the chemical reaction (DGo' is negative in the direction of ATP hydrolysis. The magnitude of DG depends also on concentrations of ATP, ADP, and Pi .) Two examples of such coupling are: 1. Active transport. Spontaneous ATP hydrolysis (negative DG) is coupled to (drives) ion flux against a gradient (positive DG). For an example, see the discussion of SERCA.