Succinic dehydrogenase active sites

The enzyme succinate dehydrogenase (SDH) is competitively inhibited by malo-nate. Figure 14.14 shows the structures of succinate and malonate. The structural similarity between them is obvious and is the basis of malonate s ability to mimic succinate and bind at the active site of SDH. However, unlike succinate, which is oxidized by SDH to form fumarate, malonate cannot lose two hydrogens consequently, it is unreactive. 

Studies of protein film electrocatalysis have also been illuminating. For example, succinate dehydrogenase displays an unusual optimal potential for activity. The enzyme contains four redox sites a flavin, a... 

Fe 2S], a [4Fe-4S] and a [3Fe-4S] center. The enzyme catalyzes the reversible redox conversion of succinate to fumarate. Voltammetry of the enzyme on PGE electrodes in the presence of fumarate shows a catalytic wave for the reduction of fumarate to succinate (much more current than could be accounted for by the stoichiometric reduction of the protein active sites). Typical catalytic waves have a sigmoidal shape at a rotating disk electrode, but in the case of succinate dehydrogenase the catalytic wave shows a definite peak. This window of optimal potential for electrocatalysis seems to be a consequence of having multiple redox sites within the enzyme. Similar results were obtained with DMSO reductase, which contains a Mo-bis(pterin) active site and four [4Fe 4S] centers. 

Mitochondrial succinate dehydrogenase, which catalyzes the reaction of Eq. 15-21, contains a flavin prosthetic group that is covalently attached to a histidine side chain. This modified FAD was isolated and identified as 8a-(Ne2-histidyl)-FAD 219 The same prosthetic group has also been found in several other dehydrogenases.220 It was the first identified member of a series of modified FAD or riboflavin 5 -phosphate derivatives that are attached by covalent bonds to the active sites of more than 20 different enzymes.219... 

At very high substrate concentrations deviations from the classical Michaelis-Menten rate law are observed. In this situation, the initial rate of a reaction increases with increasing substrate concentration until a limit is reached, after which the rate declines with increasing concentration. Substrate inhibition can cause such deviations when two molecules of substrate bind immediately, giving a catalytically inactive form. For example, with succinate dehydrogenase at very high concentrations of the succinate substrate, it is possible for two molecules of substrate to bind to the active site and this results in non-functional complexes. Equation S.19 gives one form of modification of the Michaelis-Menten equation. 

A classic example of competitive inhibition is the inhibition of succinate dehydrogenase by malonate, a structural analogue of succinate. Competitive inhibitors are usually structural analogues of the substrate, the molecule with which they are competing. They bind to the active site but either do not have a structure that is conducive to enzymatic modification or do not induce the proper orientation of catalytic amino acyl residues required to affect catalysis. Consequently, they displace the substrate from the active site and thereby depress the velocity of the reaction. Increasing [S] will displace the inhibitor. 

At the active site of beef heart mitochondrial succinate dehydrogenase (EC 1.3.99.1), the FAD is covalently linked by C-8a to nitrogen of a histidine residue (Fig. 2b) [20]. In the catalytic reaction, removal of a proton from C-3 of the succinate may be followed by attack of the 3-carbanion on N-5 of the FAD to form an intermediate adduct, which breaks down with loss of a proton from C-2 of the succinate, giving fumarate and a reduced FAD moiety [21]. This mechanism is not certain, but it is established that the succinate loses two non-equivalent hydrogen atoms by a trans elimination (Fig. 4) [22], In other enzymes, different types of covalent attachment of the FAD are known [23]. 

The [2Fe 2S], [3Fe S], and [4Fe S] clusters that are found in simple Fe S proteins are also constituents of respiratory and photosynthetic electron transport chains. Multicluster Fe S enzymes such as hydrogenase, formate dehydrogenase, NADH dehydrogenase, and succinate dehydrogenase feed electrons into respiratory chains, while others such as nitrate reductase, fhmarate reductase, DMSO reductase, and HDR catalyze the terminal step in anaerobic electron transport chains that utihze nitrate, fumarate, DMSO, and the CoB S S CoM heterodisulfide as the respiratory oxidant. All comprise membrane anchor polypeptide(s) and soluble subunits on the membrane surface that mediate electron transfer to or from Mo cofactor (Moco), NiFe, Fe-S cluster or flavin active sites. Multiple Fe-S clusters define electron transport pathways between the active site and the electron donor or... 

The mitochondrial respiratory chain, which contains at least 13 Fe-S clusters (Figure 6), perhaps best illustrates the importance of Fe-S clusters in membrane-bound electron transport. Electrons enter via three principal pathways, from the oxidation of NADH to NAD+ (NADH-ubiquinone oxidoreductase or Complex I) and succinate to fumarate (succinate ubiquinone oxidoreductase or Complex II), and from the /3-oxidation of fatty acids via the electron transferring flavoprotein (ETF-ubiquinone oxidoreductase). All three pathways involve a complex Fe S flavoprotein dehydrogenase, that is, NADH dehydrogenase, succinate dehydrogenase, and ETF dehydrogenase, and in each case the Fe-S clusters mediate electron transfer from the flavin active site to the ubiquinone pool via protein-associated ubiquinone. 

Flavocytochrome Fumarate Reductase. The flavocytochrome fumarate reductase (Fff) is a soluble periplasmic protein from Shewanella spp. that reduces fumarate but does not oxidize succinate, in contrast to the membrane-bound fumarate reductases that are related to succinate dehydrogenases, and transfer electrons from quinol to fumarate. It is a monomeric protein of 63.8 kDa that is composed of three domains. The N-terminal domain contains four c-type hemes, and the flavin domain contains noncovalently bound FAD and is related to flavoprotein subunits of membrane-bound fumarate reductases and succinate dehydrogenases. There is also a third domain in the flavocytochromes that has considerable flexibility and may be involved in controlling access of substrate to the active site. The macroscopic redox potentials of the fom hemes of Ffr are —102, —146, —196, and -23 8 mV, while that of FAD is —152 mV. The low redox potential of FAD in Ffr compared to that in membrane-bound fumarate reductase (—55 mV) may explain why it is unable to oxidize succinate. 

During the terminal stages of electron transfer in complex II, cytochrome bysg is involved however, its specific function is not understood. Oxaloacetate and malonate are competitive inhibitors of succinate dehydrogenase and compete with the substrate for binding at the active site (Chapters 6 and 13). Carboxin and thenoyltrifluoroacetone (Figure 14-7) inhibit electron transfer from FADH2 to CoQ. 

Although enzymes catalyze only certain reactions or certain types of reaction, they are still subject to interference. When the activated complex is formed, the substrate is adsorbed at an active site on the enzyme. Other substances of similar size and shape may be adsorbed at the active site. Although adsorbed, they will not undergo any transformation. However, they do compete with the substrate for the active sites and slow down the rate of the catalyzed reaction. This is called competitive inhibition. For example, the enzyme succinic dehydrogenase will specifically catalyze the dehydrogenation of succinic acid to form fumaric acid. But other compounds similar to succinic acid will competitively inhibit the reaction. Examples are other diprotic acids such as malonic and oxalic acids. Competitive inhibition can be reduced by increasing the concentration of the substrate relative to that of the interferent so that the majority of enzyme molecules combine with the substrate. 

A competitive inhibitor binds to the active site of an enzyme and thus competes with snbstrate molecnles for the active site. Competitive inhibitors often have molecular structures that are similar to the normal substrate of the enzyme. The competitive inhibition of snccinate dehydrogenase by malonate is a classic example. Succinate dehydrogenase catalyzes the oxidation of the substrate succinate to form fumarate by transferring two hydrogens to the coenzyme FAD ... 

Malonate, having a structure similar to succinate, competes for the active site of succinate dehydrogenase and thus inhibits the enzyme ... 

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