|Lecture Notes: 21 April||
Given the requirement for a very tight inner-mitochondrial membrane in order to maintain the proton electrochemical gradient, how do important charged molecules such as ATP and NADH get across the membrane?
ADP & ATP are obviously among the most important substances to transport into and out of the mitochondria. Adenine nucleotide translocase exchanges matrix ATP for cytosolic ADP in their magnesium-free forms. Note that in this exchange ATP4- leaves the matrix as ADP3- goes in, resulting in a net loss of (1-) for the matrix. This increases the charge gradient across the membrane, and thus must be driven by the mitochondrial proton gradient. Of course Pi must also be transported across the membrane with ADP to make ATP in the matrix. This is accomplished using another transporter which co-transports a dihydrogen phosphate and a single proton in an electroneutral process. Note the addition of these two processes is equivalent to moving one proton from the cytosol to the matrix, costing the gradient one proton (moving a negative charge out is equivalent to moving a positive charge in).
The net cost of providing an ATP to the cytosol is thus four protons: three to convert ADP + Pi to ATP and one to transport ATP out of, while bringing ADP and Pi into, the matrix. This accounts for the theoretical yield of ATP: (10 H+/NADH)/(4 H+/ATP) = 2.5 ATP/NADH. (Note that this means that bacteria may get more ATP (up to 38 ATP's instead of the 32 expected in mammals).
Reducing Equivalent Shuttles: In aerobic metabolism NADH from glycolysis must be regenerated to NAD+ in the mitochondria. Two shuttles are important in different tissues/organisms for this process:
The glycerol-3-P is then reoxidized to dihydroxyacetone-P by Flavoprotein dehydrogenase. This enzyme is situated on the inner mitochondrial membranes outer surface. It uses FAD to oxidize the glycerol-3-P to dihydroxyacetone-P, passing the electrons to CoQ (note the similarity to succinate DH, except for the location on the outer instead of the inner surface of the membrane). The organism can thus get 1.5 ATP equivalents for this NADH.
The ETS appears to be regulated largely by the availability of ADP and NADH. For most catabolic situations [ADP] will be the controlling factor. Note that the effects of [ADP] will integrate the regulation of TCA and Glycolysis with that of ETS.
Fats come from two main sources: stored body fat and dietary fat. Dietary fat must first be emulsified to increase its surface area for contact with the water soluble lipases. This occurs largely in the duodenum after mixing with the bile acids, a family of cholesterol derived detergents. Triacylglycerols can then be hydrolyzed by pancreatic lipase to free fatty acids and 2-monoacylglycerol:
The fatty acids and monoacylglycerol are absorbed by the intestinal cells, converted to fatty acyl CoA and reassembled into triacylglycerols. The triacyl glycerols then assemble with phospholipids and lipoproteins to form chylomicrons for transport through the lymph and blood to the tissues.
When the chylomicrons reach tissue cells the triacylglycerols are again hydrolyzed by lipoprotein lipase to fatty acids which can be taken up by the peripheral tissue cells. In adipose cells the fatty acids are then converted into fatty acyl CoA's and combined into triacylglycerols for storage. Alternatively the fatty acids can be broken down for energy using the beta-oxidation pathway.
Free fatty acids are introduced into the cytosol, but -oxidation occurs in the mitosol. Two situations occur.
Short to medium length fatty acids are permeable to the mitochondrial membrane. They are activated to fatty acyl CoA derivatives in the mitochondrial matrix by Butyryl-CoA Synthetase:
Note that two ATP equivalents are required: the phosphoanhydride and thioester bonds are of similar free energies, so a second phosphoanhydride bond is also hydrolyzed to drive the reaction to completion.
Long chain fatty acids are bound to Fatty acid binding protein for transport within the cytosol. They are impermeable to the inner mitochondrial membrane (they are also toxic to the mito!). They are thus esterified in the cytosol by microsomal Fatty acyl CoA synthetase in a reaction identical to the one shown above. Again the reaction is driven by the hydrolysis of pyrophosphate. The enzyme involves an acyl AMP intermediate:
with Ping Pong Bi Uni-Uni Bi kinetics:
Carnitine Carrier: The resulting acyl CoA ester is still not permeable to the mitochondrial membrane so a carrier system is needed. In this system the fatty acyl group is transferred from CoA-S to carnitine, diffuses across the membrane, and then transferred back to another CoA-S within the matrix:
The carnitine transport step across the inner membrane is the slow step and flux generating step for -oxidation of long chain fatty acids. Note that this system maintains separate pools of CoASH in the cytosol vs. the matrix.
Last modified 6 May 2010