Question

1. How does malonate affect the initial velocity of succinate dehydrogenase?

2. Explain why initial velocity varies directly with enzyme concentration, and how this related to the Michaelis Menten equation

3. How does malonate affect the initial velocity of an reaction?

4. What feature(s) of malonate makes it a competitive inhibitor of SDH?

Answer 1

Ans(a):- The kinetic behaviour of succinate dehydrogenase [EC 1.3.99.1] in three fibre types of rat gastrocnemius was examined by a quantitative histochemical method without disruption of the cellular structure. 2-(2-Benzothiazolyl)-3-(4-phthalhydrazidyl)-5-styryl-tetrazolium chloride (BPST) and phenazine methosulphate were used as electron acceptors. On measurement of the absorbance value at 530 nm of BPST formazan, produced by the succinate dehydrogenase reaction in sections, it was found that the staining intensity of succinate dehydrogenase was linearly proportional to both the incubation time and the thickness of the slice therefore, the initial velocity of the staining could be calculated. By Michaelis-Menten (1913) treatment of the dependence of the initial velocity on the substrate concentration in the absence and the presence of a competitive inhibitor, malonate, the Km andVmax values for succinate and the Ki value for malonate were obtained. The Km and Ki values of the three fibre types were similar. The ratio of theVmax values of type A, B and C fibres was 1.0∶2.0∶3.3. The temperature dependence of the kinetic parameters was very similar in the three fibre types. These findings confirm that the differences in the staining intensity of the three fibre types reflect differences in the amounts, but not the properties, of succinate dehydrogenase.

Malonate is a competitive inhibitor of the enzyme succinate dehydrogenase: malonate binds to the active site of the enzyme without reacting, and so competes with succinate, the usual substrate of the enzyme. ... The chemical malonate decreases cellular respiration.

uccinate dehydrogenase (SDH) or succinate-coenzyme Q reductase (SQR) or respiratory Complex II is an enzyme complex, found in many bacterial cells and in the inner mitochondrial membrane of eukaryotes. It is the only enzyme that participates in both the citric acid cycle and the electron transport chain.

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)

Ans(b):

Enzymes are protein molecules that act as biological catalysts by increasing the rate of reactions without changing the overall process. They are long chain amino acids bound together by peptide bonds. Enzymes are seen in all living cells and controlling the metabolic processes in which they converted nutrients into energy and new cells. Enzymes also help in the breakdown of food materials into its simplest form. The reactants of enzyme catalyzed reactions are termed as substrates. Each enzyme is quite specific in character, acting on a particular substrates to produce a particular products. The central approach for studying the mechanism of an enzyme-catalyzed reaction is to determine the rate of the reaction and its changes in response with the changes in parameters such as substrate concentration, enzyme concentration, pH, temperature etc .This is known as enzyme kinetics.

One of the important parameters affecting the rate of a reaction catalyzed by an enzyme is the substrate concentration, [S]. During enzyme substrate reaction, the initial velocity V0 gradually increases with increasing concentration of the substrate. Finally a point is reached, beyond which the increase in V0 will not depend on the [S]. When we plot a graph with substrate concentration on the X axis and corresponding velocity on Y axis. It can be observed from the graph that as the concentration of the substrate increases, there is a corresponding increase in the V0. However beyond a particular substrate concentration, the velocity remains constant without any further increase. This maximum velocity of an enzyme catalysed reaction under substrate saturation is called the Vmax , Maximum velocity.

                           

Michaelis – Menten Equation

Leonor Michaelis and Maud Menten postulated that the enzyme first combines reversibly with its substrate to form an enzyme-substrate complex in a relatively fast reversible step:

                                       Eqn.1

In the next step, this ES complex is breaks down in to the free enzyme and the reaction product P:

                                                Eqn.2

Since the second step is the rate limiting step, the rate of overall reaction must be proportional to the concentration of the ES that reacts in the second step. The relationship between substrate concentration, [S] and Initial velocity of enzyme, V0 (Fig. 1) has the same general shape for most enzymes (it approaches a rectangular hyperbola). This can be expressed algebraically by the Michaelis-Menten equation. Based on their basic hypothesis that the rate limiting step in enzymatic reactions is the breakdown of the ES complex to free enzyme and product, Michaelis and Menten derived an equation which is;

                                   Eqn.3

The necessary terms in this reaction are [S], V0, Vmax, and Km (Michaelis constant),. All these terms can be   measured experimentally.

Lineweaver – Burke plot

In 1934, Lineweaver and Burke made a simple mathematical alteration in the process by plotting a double inverse of substrate concentration and reaction rate.

                                             Eqn.4

For enzymes obeying the Michaelis-Menten relationship, the “double reciprocal” of the V0 versus [S] from the first graph,(fig1) yields a straight line (Fig. 2). The slope of this straight line is KM /Vmax, which has an intercept of 1/Vmax on the 1/V0 axis, and an intercept of -1/KM on the 1/[S] axis. The double-reciprocal presentation, also called a Lineweaver-Burk plot. The main advantage of Lineweaver-Burk plot is to determine the Vmax more accurately, which can only be approximated from a simple graph of V0 versus [S] (Fig 1).

   
                                           Fig2: Lineweaver-Burk plot.

Adapted from David L. Nelson, Michael M. Cox , Lehninger principles of biochemistry, 4th edition.

Principle:

The enzyme α Amylase can catalyze the hydrolysis of internal α -1,4-glycosidic bond present in starch with the production of reducing sugars. In the study of substrate concentration on enzyme kinetics, the enzyme is kept constant where as the concentration of Starch is taken in increasing order. As the substrate concentration increases, the amount of products produced in every successive tube also increases. This was explained by Michealis and others that an enzyme catalyzed reaction at varying substrate concentrations is diphasic i.e. at low substrate concentration the active sites on molecules (enzyme) are not occupied by substrate and the enzyme rate varies with substrate molecules concentration (phase1). As the number of substrate molecules increases, the enzyme attains the saturation level, since there is no more reaction sites remaining for binding. So the enzyme can work with full capacity and its reaction rate is independent of substrate concentration. (Phase II).

This Enzyme – substrate reaction can be determined by measuring the increase in reducing sugars using the 3, 5 Dinitro salycilic acid reagent. In an alkaline condition, the pale yellow colored the 3, 5- dinitro salicylic acid undergo reduction to yield orange colored 3- amino -5-nitrosalicylic acid. The absorbance of resultant solutions is read at 540nm. The intensity of color depends on the concentration of reducing sugars produced.

                                  α Amylase
                    Starch     Maltose + glucose

      

Ans(d):- Malonate is a competitive inhibitor of the enzyme succinate dehydrogenase: malonate binds to the active site of the enzyme without reacting, and so competes with succinate, the usual substrate of the enzyme.

Malonate is a competitive inhibitor, which means it binds to the active site of SDH, blocking the substrate succinate from binding to it. The presence of Malonate interrupts the Krebs Cycle, and the formation of FADH is prevented

Enzymes can be activated or inhibited in different ways. Aspecific inhibition is possible by a drastic change in pH, temperature or ionic concentration. Much more information can be gained by using specific enzyme inhibitors. Such enzyme kinetic analysis may shed light on the mechanism of enzyme activity. Competitive inhibitor binds reversibly to the free enzyme, the enzyme-inhibitor complex has no catalytic activity and thus less active enzyme is available for interaction with the substrate. This effect is reflected in the increase of the apparent Michaelis constant (Km), because more substrate is required to achieve the half-maximal reaction rate. At extremely high (S>>Km) substrate concentration the inhibition becomes negligible, the Vmax is not changed. Either the substrate or the inhibitor is bound to the enzyme, not both of them.

On the basis of the aforementioned definition we can see that competitive inhibition is only a characteristic of kinetic behaviour of the enzyme, but does not say anything about the structural background of the interaction of inhibitor and enzyme, about the binding site of the inhibitor, which can be either the active site or an allosteric site. Historically the first described competitive inhibitors were structural analogues of the substrates that bind to the active site instead of the substrate, Malonate used in the present experiment is also a substrate analogue.

Competitive inhibition is a form of enzyme inhibition where binding of the inhibitor to the active site on the enzyme prevents binding of the substrate and vice versa. Malonate is a competitive inhibitor of the enzyme succinate dehydrogenase: malonate binds to the active site of the enzyme without reacting, and so competes with succinate, the usual substrate of the enzyme.