The n [subscript H] value obtained from an analysis of the dependence of initial velocity oxaloacetate synthesis on acetyl CoA concentration using the Hill equation was shown to depend on the fixed concentrations of pyruvate and bicarbonate in the assay solutions. The time period over which the reaction rate was linear with time was found to depend on the acetyl CoA concentration in the assay solution. At high concentrations of acetyl CoA, the process of dilution inactivation was prevented, and hence the reaction was linear with time for a longer period than at low concentrations of acetyl CoA.
Dilution inactivation was shown not to involve formation of catalytically active pyruvate carboxylase dimers or monomers. When an experiment was designed in which all substrates and activators were present at saturating levels regardless of the acetyl CoA concentration, and the enzyme concentration was raised to a level where dilution inactivation did not occur, the dependence of the rate of oxaloacetate synthesis on acetyl CoA concentration was found to be consistent with lack of co-operativity of binding of acetyl CoA to pyruvate carboxylase.
Thesis Ph. This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. They are effective in the treatment of diphtheria, gonorrhea, pneumonia, syphilis, many pus infections, and certain types of boils. Penicillin G was the earliest penicillin to be used on a wide scale. However, it cannot be administered orally because it is quite unstable; the acidic pH of the stomach converts it to an inactive derivative.
The major oral penicillins—penicillin V, ampicillin, and amoxicillin—on the other hand, are acid stable. Some strains of bacteria become resistant to penicillin through a mutation that allows them to synthesize an enzyme—penicillinase—that breaks the antibiotic down by cleavage of the amide linkage in the lactam ring.
To combat these strains, scientists have synthesized penicillin analogs such as methicillin that are not inactivated by penicillinase. Their allergic reaction can be so severe that a fatal coma may occur if penicillin is inadvertently administered to them. Fortunately, several other antibiotics have been discovered. Most, including aureomycin and streptomycin, are the products of microbial synthesis. Others, such as the semisynthetic penicillins and tetracyclines, are made by chemical modifications of antibiotics; and some, like chloramphenicol, are manufactured entirely by chemical synthesis.
They are as effective as penicillin in destroying infectious microorganisms. Many of these antibiotics exert their effects by blocking protein synthesis in microorganisms. Initially, antibiotics were considered miracle drugs, substantially reducing the number of deaths from blood poisoning, pneumonia, and other infectious diseases.
Some seven decades ago, a person with a major infection almost always died. Today, such deaths are rare. Seven decades ago, pneumonia was a dreaded killer of people of all ages.
Today, it kills only the very old or those ill from other causes. Antibiotics have indeed worked miracles in our time, but even miracle drugs have limitations. Not long after the drugs were first used, disease organisms began to develop strains resistant to them. In a race to stay ahead of resistant bacterial strains, scientists continue to seek new antibiotics.
The penicillins have now been partially displaced by related compounds, such as the cephalosporins and vancomycin. Unfortunately, some strains of bacteria have already shown resistance to these antibiotics. This enzyme is absent in mammals. Pyruvate carboxylase is homotetramer. The lysine residue of active site is covalently attached with biotin. Pyruvate carboxylase catalyzes the addition of CO 2 to pyruvate to form oxaloacetate. The activated CO 2 is then transferred to pyruvate to form the carboxyl group of Oxaloacetate.
Pyruvate carboxylase is activated by acetyl CoA and inhibited by high concentrations of many acyl CoA derivatives. As the concentration of oxaloacetate is depleted through the efflux of TCA cycle intermediates, the rate of the citrate synthase reaction decreases and acetyl CoA concentration rises.
The acetyl CoA then activates pyruvate carboxylase to synthesize more oxaloacetate. Citrate synthase reaction Citrate synthase is controlled by the concentration of acetyl-CoA which is, in turn, governed by the activity of pyruvate dehydrogenase complex.
FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate [ 9 ]. The covalent bond between FAD and succinate dehydrogenase. The 2Fe-2S cluster of succinate dehydrogenase [ 9 ]. The hydrophobic anchoring subunits are integral membrane proteins and interact with quinone substrates. The yeast and mammalian SDH also contains a b -type heme.
Oyedotun et al. Together, the Fp and Ip form a catalytic dimer that is attached to the membrane by the anchoring subunits, thereby composing the holoenzyme. The SDH subunits are translated in the cytoplasm, targeted to mitochondria by cleavable amino-terminal presequences, translocated across both mitochondrial membranes, and finally assembled with each other and their respective co-factors into a functional complex [ 10 ]. The quaternary structure model of the SDH for different cells, e.
First subunit of SDH provides the binding site for the oxidation of succinate. The side chains Thr, His, and Arg stabilize the molecule while FAD oxidizes and carries the electrons to the first of the iron-sulfur [2Fe-2S] clusters.
Whereas, ubiquinone binding site is located is in a gap composed of three SDH subunits. Ubiquinone is stabilized by the side chains of His of second subunit, Ser27 and Arg31 of third subunit C, and Tyr83 of fourth subunit.
The quinine ring is surrounded by Ile28 of third subunit and Pro of second subunit B. These residues, along with Il, Trp, and Trp of second subunit B, and Ser27 C atom of third subunit, form the hydrophobic environment of the quinine binding pocket. The succinate binding site and ubiquinone binding site are connected by a chain of redox centres including FAD and the iron-sulfur clusters.
In the place for heme b , the N 2 atom of Sdh3p His and the S atom of Sdh4p Cys78 are correctly oriented to form coordinating bonds with the central iron atom of the heme. The distance between the iron atom and the N 2 atom is 2.
UQ can be docked into two spatially separated sites with an edge-to-edge distance of The new amino acid residues that may determine the structural or catalytic properties of each of the two quinone binding sites were identified. The model also provided insight into the unusual use of a cysteine Sdh4p Cys78 as the second heme ligand instead of the histidine residues [ 10 ]. Succinate dehydrogenase is a key enzyme in intermediary metabolism and aerobic energy production in living cells.
This enzymes catalyses the oxidation of succinate into fumarate in the Krebs cycle 1 , derived electrons being fed to the respiratory chain complex III to reduce oxygen and form water 2. This builds up an electrochemical gradient across the mitochondrial inner membrane allowing for the synthesis of ATP. Alternatively, electrons can be diverted to reduce the ubiquinone pool UQ pool and provide reducing equivalents necessary to reduce superoxide anions originating either from an exogenous source or from the respiratory chain itself 3 [ 13 ] Figure 4.
The functions of the succinate dehydrogenase in the mitochondria [ 13 ]. In the reaction of oxidation of succinate to fumarate, two hydrogen atoms are removed from substrate by flavin adenine dinucleotide FAD , a prosthetic group that is tightly attached to succinate dehydrogenase Figure 5. The succinate dehydrogenase reaction.
Ubiquinone is then reduced to ubiquinol QH2. The generation of adenosine triphosphate ATP in mitochondria is coupled to the oxidation of nicotinamide adenine dinucleotide NADH and FADH2 and reduction of oxygen to water within the respiratory chain and a three-dimensional structure of the mitochondrial respiratory membrane protein complex II.
The substrate analog malonate is a competitive inhibitor of the succinate dehydrogenase complex. Malonate, like succinate, is a dicarboxylate that binds to cationic amino acid residues in the active site of the succinate dehydrogenase complex. However, malonate cannot undergo oxidation because it lacks the -CH2 - CH2- group necessary for dehydration. To study the effects of a competitive inhibitior on the activity of succinate dehydrogenase, malonate will be added to a reaction mixture; malonate is sufficiently different from succinate that it cannot de dehydrogenated, i.
SDH is a difficult enzyme to extract from respiratory membrane whilst still retaining its in vivo properties. Most of the extraction procedures used in early work were rather drastic and yielded soluble preparations of rather dubious integrity.
However, the recent introduction of a more gentle method, involving disruption of the membrane with chemotropic agents, has yielded an active and nearly homogeneous enzyme of relatively low molecular weight 97, This enzyme can be separated by freezing and thawing into two inactive subunits. One of these, an iron sulphur flavoprotein of molecular weight 70,, contains one mole of FAD and four moles each of iron and labile sulphide per mole of protein; The other, an iron-sulphur protein of molecular weight 27,, also contains four moles each of iron and labile sulphide.
It was determined that the large subunit of SDH is essential for catalytic activity, but the function of the small subunit, be it catalytic or regulatory [ 8 ]. Electronic paramagnetic resonance measurements of SDH components indicated that at least three separate centres are present.
The redox potential of S-2 is rather too low to allow this centre to be catalytically active in the forward direction [ 8 , 15 ]. For example, phosphorylation of the Sdh1 subunit leads to attenuate activity of SDH. The activity of this enzyme may be also modulated by Krebs cycle intermediates including oxaloacetate or malonate which are strong inhibitors. Mechanisms of inhibition by these compounds differ significantly because oxaloacetate, a competitive inhibitor of succinate dehydrogenase, bounds with a sulfhydryl group of the enzyme to abolish the enzyme activity [ 16 ].
It is known that SDH is sensitive to different thiol-binding reagents. Inhibition of the enzyme by these kinds of reagents resulted from the modification of a sulfhydryl group located at the active site.
This thiol, although not essential for substrate binding or catalysis, could influence the binding of dicarboxylates, probably by steric hindrance when a larger group or a charged group were attached to it. The inhibition of SDH by histidine specific reagents was also reported, and the participation of an imidazole ring in the initial step of succinate oxidation was suggested. The inactivation of SDH by phenylglyoxal and 2,3 —butanedione showed the presence of an arginine-residues that interacts with dicarboxylate to form the primary enzyme-substrate complex [ 17 ].
SDH is not only known to catalyse a unique reaction, which requires the participation of its four subunits, but deleterious mutations in any of the SDH genes should invariably result in a decreased SDH activity. Therefore, the striking phenotypic differences associated with mutations in the four subunits raise puzzling questions.
SDH also plays a specific role in the maintenance of the mitochondrial UQ pool reduction. Ubiquinone, beside its function as an electron carrier mediating electron transfer, is admittedly working as a powerful antioxidant in biological membranes. Then, only a portion of the UQ pool may be actually involved in electron transfer depending on dehydrogenases involved. Accordingly, the measurable redox status of the UQ pool should result from the reducing activity of the different dehydrogenases, the oxidising activity of complex III and the kinetic equilibrium in the pool.
The UQ pool therefore represents an electron sink and, when reduced, an antioxidant reservoir in the mitochondrial inner membrane. However, UQ is a double-faced compound, possibly working as either an antioxidant when fully reduced to ubiquinol, or a pro-oxidant when semi-reduced to the unstable ubisemiquinone form.
Possibly together with reduced cytochrome b, semi-reduced quinones constitute the prominent source of superoxides. Finally, when defective, the abnormal amount of superoxides can be produced, e. Delivering electrons for the full reduction of UQ to UQH2 might then be of a tremendous importance for the control of oxygen toxicity in the mitochondria.
Therefore, the SDH, thanks to its unique redox properties, may be a key enzyme to control UQ pool redox poise under these conditions [ 13 ]. Disruption of complex II activity should alter TCA cycle metabolite levels in the mitochondrial matrix. The succinate is the most efficient energy source, so the SDH activity assay can be an important method for measurement of the yeast vitality in scope to control, e. SDH activities can be measured in vitro in cell lysates or in mitochondrial fraction as well as in situ in individual cells.
Since SDH is bound to the inner membrane, it is easily isolated along with the mitochondria by different techniques: sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method [ 20 ]. The mitochondrial fraction is the source of the enzyme. To use an artificial electron acceptor, the normal path of electrons through the mitochondrial electron transport system must be blocked. This is accomplished by adding either sodium azide or potassium cyanide to the reaction mixture.
These poisons inhibit the transfer of electrons from cytochrome a3 to the final electron acceptor, oxygen, thus electrons cannot be passed along by the preceding cytochromes and coenzyme Q. The reduction of DCIP can be followed spectrophotometrically since the oxidized form of the dye is blue and the reduced form is colorless. This reaction can be summarized as. The change in absorbance, measured at nm, can be used to follow the reaction over time [ 21 ].
To use an artificial electron acceptor, the normal path of electrons in the electron transport chain must be blocked. This is accomplished by adding either potassium cyanide or sodium azide to the reaction mixture. The rate of the disappearance of the blue color is proportional to the concentration of enzyme.
The change in absorbance of the mixture is measured as a function of time and the enzyme concentration is determined from these data.
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