Matrix dehydrogenases

Figure 11.6 Schematic representation of Ca2+ transport in and out of mitochondria, showing all the Ca2+ transporters and activation of matrix dehydrogenases. PTP—permeability transition pore. (From Carafoli, 2003. Copyright 2003, with permission from Elsevier.)...

According to the chemiosmotic coupling hypothesis, ATP synthesis decreases the proton electrochemical gradient and hence stimulates the respiratory chain to pump more protons across the mitochondrial inner membrane and maintain the gradient. However, electron supply to the respiratory chain also affects respiration and ATP synthesis. For example, calcium stimulates mitochondrial matrix dehydrogenase, and increases the electron supply to the respiratory chain and hence the rate of respiration and ATP synthesis. 

Effect of Various Activating Systems on the Structure-Linked Latency of Lysosomal Hydrolases and Mitochondrial Matrix Dehydrogenases... 

Recent studies on mitochondrial structure have demonstrated the localization of mitochondrial enzymes in two membranes and two potential spaces (Fig. 4). For a substrate to react with an enzyme located in the mitochondrial matrix, it must first penetrate both outer and inner membranes. The outer membrane does not appear to hinder the passage of small molecules and substrates. The inner mitochondrial membrane, more analogous to the lysosomal membrane, is relatively impermeable to small molecules and ions (Chappell and Crofts, 1966). For anionic substrates to reach the matrix dehydrogenases, specific transporting systems are present in the inner mitochondrial membrane. Even for diose enzymes located in the inner membrane, penetration of that structure must occur as the active center of such enzymes is only available from the matrix side of the membrane (Chappell, 1968). Such data demonstrate that the presence of a membrane does not in itself induce enzyme latency as creatinine kinase and adenylate kinase localized in the intermembrane space, do not show latency. Moreover, monoamine oxidase and NADH cytochrome c reductase, present in the outer mitochondrial membrane, do not show latency. 

The space inside the inner mitochondrial membrane is called the matrix, and it contains most of the enzymes of the TCA cycle and fatty acid oxidation. (An important exception, succinate dehydrogenase of the TCA cycle, is located in the inner membrane itself.) In addition, mitochondria contain circular DNA molecules, along with ribosomes and the enzymes required to synthesize proteins coded within the mitochondrial genome. Although some of the mitochondrial proteins are made this way, most are encoded by nuclear DNA and synthesized by cytosolic ribosomes. 

This is a crucial point because (as we will see) proton transport is coupled with ATP synthesis. Oxidation of one FADHg in the electron transport chain results in synthesis of approximately two molecules of ATP, compared with the approximately three ATPs produced by the oxidation of one NADH. Other enzymes can also supply electrons to UQ, including mitochondrial 5w-glyc-erophosphate dehydrogenase, an inner membrane-bound shuttle enzyme, and the fatty acyl-CoA dehydrogenases, three soluble matrix enzymes involved in fatty acid oxidation (Figure 21.7 also see Chapter 24). The path of electrons from succinate to UQ is shown in Figure 21.8. 

Most of the NADH used in electron transport is produced in the mitochondrial matrix space, an appropriate site because NADH is oxidized by Complex I on the matrix side of the inner membrane. Furthermore, the inner mitochondrial membrane is impermeable to NADH. Recall, however, that NADH is produced in glycolysis by glyceraldehyde-3-P dehydrogenase in the cytosol. If this NADH were not oxidized to regenerate NAD, the glycolytic pathway would cease to function due to NAD limitation. Eukaryotic cells have a number of shuttle systems that harvest the electrons of cytosolic NADH for delivery to mitochondria without actually transporting NADH across the inner membrane (Figures 21.33 and 21.34). 

In the glycerophosphate shuttle, two different glycerophosphate dehydrogenases, one in the cytoplasm and one on the outer face of the mitochondrial inner membrane, work together to carry electrons into the mitochondrial matrix (Figure 21.32). NADH produced in the cytosol transfers its electrons to dihydroxyaeetone phosphate, thus reducing it to glyeerol-3-phosphate. This metabolite is reoxidized by the FAD -dependent mitochondrial membrane enzyme to... 

The second electron shuttle system, called the malate-aspartate shuttle, is shown in Figure 21.34. Oxaloacetate is reduced in the cytosol, acquiring the electrons of NADH (which is oxidized to NAD ). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD to NADH in the matrix. This mitochondrial NADH readily enters the electron transport chain. The oxaloacetate produced in this reaction cannot cross the inner membrane and must be transaminated to form aspartate, which can be transported across the membrane to the cytosolic side. Transamination in the cytosol recycles aspartate back to oxaloacetate. In contrast to the glycerol phosphate shuttle, the malate-aspartate cycle is reversible, and it operates as shown in Figure 21.34 only if the NADH/NAD ratio in the cytosol is higher than the ratio in the matrix. Because this shuttle produces NADH in the matrix, the full 2.5 ATPs per NADH are recovered. 

Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in /3-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give (i-hydroxy-(i-methyl-glutaryl-CoA, commonly abbreviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 25. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, /3-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to /3-hydroxybutyrate. 

The acetyl-CoA derived from amino acid degradation is normally insufficient for fatty acid biosynthesis, and the acetyl-CoA produced by pyruvate dehydrogenase and by fatty acid oxidation cannot cross the mitochondrial membrane to participate directly in fatty acid synthesis. Instead, acetyl-CoA is linked with oxaloacetate to form citrate, which is transported from the mitochondrial matrix to the cytosol (Figure 25.1). Here it can be converted back into acetyl-CoA and oxaloacetate by ATP-citrate lyase. In this manner, mitochondrial acetyl-CoA becomes the substrate for cytosolic fatty acid synthesis. (Oxaloacetate returns to the mitochondria in the form of either pyruvate or malate, which is then reconverted to acetyl-CoA and oxaloacetate, respectively.)... 

The fact that adenosine and its derivatives are azo coupling components is used for immobilizing nicotinamide-adenine nucleotide (NAD+) for affinity chromatography purposes. In 12.58 NAD+ is bonded to a matrix through an azo bond. Compound 12.58 is used for the purification of dehydrogenase enzymes (Hocking and Harris, 1973). 

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

Many metabolic fuels are oxidized in the mitochondrial matrix. Pyruvate is oxidatively decarboxylated to acetyl-CoA by the pyruvate dehydrogenase complex (PDH)... 

Enzymes 7,9, and 13 form a trifunctional protein associated with the inner face of the inner mitochondrial membrane. Very-long-chain acyl-CoA dehydrogenase is also associated with other inner mitochondrial membranes while the other enzymes are in the matrix and may be loosely associated with the inner face of the inner membrane. A medium-chain 2-enoyl-CoA hydratase may also be present in the mitochondrial matrix. 

Most of the acetyl-CoA formed by 3-oxidation in liver is converted to acetoacetate by the 3-hydroxy-3-methylglutaryl-CoA pathway (Guzman and Gelen, 1993). Acetoacetate is reversibly converted to D-3-hydroxybutyrate by D-3-hy-droxybutyrate dehydrogenase in the mitochondrial matrix in all tissues. 

The oxidation of fatty acids within the Knoop-Lynen cycle occurs in the matrix. The Knoop-Lynen cycle includes four enzymes that act successively on acetyl-CoA. These are acyl-CoA dehydrogenase (FAD-dependent enzyme), enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase (NAD-dependent enzyme), and acetyl-CoA acyltrans-ferase. Each turn, or revolution, of the fatty acid spiral produces... 

It is difficult to incorporate dehydrogenases that are coupled with NAD(P) into amperometric enzyme sensors owing to the irreversible electrochemical reaction of NAD. We have developed an amperometric dehydrogenase sensor for ethanol in which NAD is electrochemically regenerated within a membrane matrix. 

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