Biochemistry - The Chemical Reactions of Living Cells Volume 2 - D. Metzler 1980

Enzymes: Protein Catalysts of Cells
Enzyme Inhibition and Activation
Isotope Exchange at Equilibrium

Let us consider the interaction of substrates A and B, which leads to The formation of products P and Q:

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If both substrates and both products are present in the system under equilibrium concentrations, the net reaction rate will be zero, yet the substrates and products will continue to interconvert through enzyme action. By introducing a small amount of a radiolabeled substrate (A* or B*) with a high specific radioactivity into the system, one can measure the rate at which the radioactive label is transferred from the labeled substrate to a specific product. In many cases, the label associated with molecule A appears in only one of the products, such as Q, whereas the label associated with molecule B appears exclusively in product P (where P is the product structurally most similar to substrate B).

FIG. 6-9. A. Dependence of the equilibrium exchange rate ($v_{eq}$) of the reaction catalyzed by Yeast hexokinase on glucose-6-phosphate concentration, maintaining a constant glucose-to-glucose-6-phosphate concentration ratio of 1/19 [37]. The reaction mixtures contained 1–2.2 mM ATP and 25.6 mM ADP (pH 6.5). B. Dependence of the equilibrium exchange rate ($v_{eq}$) of the reaction catalyzed by rabbit Skeletal Muscle Lactate dehydrogenase on lactate concentration, maintaining a constant Pyruvate-to-lactate concentration ratio of 1/35 [38]. The reaction mixtures contained 1.7 mM NAD, 30–46 $\mu$M NADH (Tris-citrate buffer, pH 7.9; 25°C).

Fig. 6-9, A shows the curves representing the dependence of the Glucose*⇄Glucose-6-phosphate exchange rate—catalyzed by hexokinase (Ch. 7, Sec. D, 6)—on the concentration of glucose-6-phosphate at a constant [Glucose]/[Glucose-6-phosphate] ratio of 1/19 (thereby keeping the substrate-to-product concentration ratio at equilibrium). As seen from the figure, the rates of this exchange reaction and the ATP⇄ADP reaction increase monotonically with increasing Substrate Concentration. This observation points to a random substrate-binding mechanism [37], while the difference in the maximum exchange rates suggests that the release of glucose-6-phosphate from the enzyme complex is presumably slower than that of ADP.

Fig. 6-9, B illustrates the corresponding dependencies for lactate dehydrogenase (based on data from [38]). In this case, as the pyruvate concentration increases, The rate of the Pyruvate*⇄Lactate exchange reaction reaches a relatively high, constant value¹⁾ (the [Pyruvate]/[Lactate] ratio is held constant at 1/35). Meanwhile, the rate of the NAD⁺⇄NADH exchange reaction first rises rapidly and then drops sharply. This behavior indicates an ordered mechanism [scheme (6-34)], in which A and Q correspond to NAD⁺ and NADH, and B and P correspond to pyruvate and lactate, respectively. At very high concentrations of B and P, the enzyme shuttles between the EA and EQ forms, and their dissociation into free enzyme and A or Q occurs very rarely, resulting in a very low A*⇄Q exchange rate.

¹⁾ The fact that the curve passes through a maximum is not considered particularly significant.

The label can also be transferred from A to P or from B to Q. As we will see below, data on such exchange reactions are extremely useful for distinguishing among the kinetic mechanisms of enzymatic reactions.