During an extended period of exercise, the enzymes involved in the glycolytic pathway in muscle tissue are actively breaking down glucose to provide the muscle energy. The liver, to maintain blood glucose levels, is synthesizing glucose via the gluconeogenic pathway.
Which of the following enzymes involved in these pathways would be most likely to exhibit Michaelis–Menten kinetics, that is, have a hyperbolic curve when plotting substrate concentration versus velocity of the reaction?
A) Fructose-1, 6-bisphosphatase
C) Lactate dehydrogenase
E) Pyruvate kinase
The correct option is C- Lactate dehydrogenase. All the above mentioned enzymes are allosteric except Lactate dehydrogenase.
In typical enzyme-catalyzed reactions, reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration. Consequently, each enzyme molecule catalyzes the conversion to product of many reactant molecules. In biochemical reactions, reactants are commonly known as substrates. The catalytic event that converts substrate to product involves the formation of a transition state, and it occurs most easily at a specific binding site on the enzyme. This site, called the catalytic site of the enzyme, has been evolutionarily structured to provide specific, high-affinity binding of substrate(s) and to provide an environment that favors the catalytic events. The complex that forms, when substrate(s) and enzyme combine, is called the enzyme substrate (ES) complex. Reaction products arise when the ES complex breaks down releasing free enzyme.
Between the binding of substrate to enzyme, and the reappearance of free enzyme and product, a series of complex events must take place. At a minimum an ES complex must be formed; this complex must pass to the transition state (ES*); and the transition state complex must advance to an enzyme product complex (EP). The latter is finally competent to dissociate to product and free enzyme. The series of events can be shown thus:
E + S <——> ES <——> ES* <——> EP <——> E + P
The kinetics of simple reactions like that above were first characterized by biochemists Michaelis and Menten. The Michaelis-Menten equation is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax and km (which are set by the particular equation). The symbols used in the Michaelis-Menten equation refer to the reaction rate [v1 or Vo], maximum reaction rate (V max), substrate concentration [S] and the Michaelis-Menten constant (km).
The Michaelis-Menten equation can be used to demonstrate that at the substrate concentration that produces exactly half of the maximum reaction rate, i.e. ½ V max the substrate concentration is numerically equal to Km. Thus, the Michaelis constant, km is the substrate concentration at which V1 is half the maximal velocity (Vmax /2) attainable at a particular concentration of enzyme (figure-1).
Figure-1- Michaelis-Menten Kinetics. A plot of the reaction velocity (V 0 ) also called Vi as a function of the substrate concentration [S] for an enzyme that obeys Michaelis-Menten kinetics. The Michaelis constant (K m) is the amount of substrate required to achieve a half maximum velocity (V max/2).
The Michaelis-Menten model cannot account for the kinetic properties of many enzymes. An important group of enzymes that do not obey Michaelis-Menten kinetics comprises the allosteric enzymes. These enzymes consist of multiple subunits and multiple active sites. Allosteric enzymes often display sigmoidal plots (Figure-2) of the reaction velocity V 0 versus substrate concentration [S], rather than the hyperbolic plots (figure-1) predicted by the Michaelis-Menten equation (equation).
Figure-2- Kinetics for an Allosteric Enzyme. Allosteric enzymes display a sigmoidal dependence of reaction velocity on substrate concentration.
In allosteric enzymes, the binding of substrate to one active site can affect the properties of other active sites in the same enzyme molecule. A possible outcome of this interaction between subunits is that the binding of substrate becomes cooperative; that is, the binding of substrate to one active site of the enzyme facilitates substrate binding to the other active sites, such cooperativity results in a sigmoidal plot of V 0 versus [S].
In addition, the activity of an allosteric enzyme may be altered by regulatory molecules that are reversibly bound to specific sites other than the catalytic sites. The catalytic properties of allosteric enzymes can thus be adjusted to meet the immediate needs of a cell. For this reason, allosteric enzymes are key regulators of metabolic pathways in the cell.
In the given condition
A) Fructose-1, 6-bisphosphatase catalyzes the conversion of Fructose 1,-6-bisphosphate to Fructose 6- Phosphate. It is an enzyme of pathway of gluconeogenesis. It overcomes the second barrier of gluconeogenesis, i.e., it reverses the reaction catalyzed by Phospho fructo kinase-1. The reaction catalyzed can be represented as follows:
It is an allosteric enzyme, Fructose 1, 6- bisphosphatase, is inhibited by AMP and activated by citrate. A high level of AMP indicates that the energy charge is low and signals the need for ATP generation (Figure-3). Conversely, high levels of ATP and citrate indicate that the energy charge is high and that biosynthetic intermediates are abundant. Under these conditions, glycolysis is nearly switched off and gluconeogenesis is promoted.
B) Hexokinase- The Hexokinase enzyme is allosterically inhibited by the product, glucose-6-phosphate.
D) Phosphofructokinase1- Phospho fructokinase is the “valve” controlling the rate of glycolysis. ATP is an allosteric inhibitor of this enzyme. AMP reverses the inhibitory action of ATP, and so the activity of the enzyme increases when the ATP/AMP ratio is lowered. Citrate inhibits phosphofructokinase by enhancing the inhibitory effect of ATP. Phosphofructokinase is also regulated by D-fructose-2, 6-bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-phosphate (figure-3).
E) Pyruvate kinase Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1, 6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine (figure-3).
Thus out of all the given options. Lactate dehydrogenase is the only enzyme which is not regulated by allosteric modification; hence it is the enzyme that would exhibit Michaelis Menten’s kinetics.
Figure-3- Reciprocal regulation of Gluconeogenesis and Glycolysis
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