PAPER No. : 16, Bio-organic and bio-physical chemistry MODULE No. :21, Bisubstrate Reactions

Transcription

1 Subject Paper No and Title Module No and Title Module Tag 16- Bio-Organic & Bio-Physical M-21 Bisubstrate Reactions CHE_P16_M21

2 TABLE OF CONTENTS 1. Learning Outcomes 2. Introduction 3. Bisubstrate reactions 3.1 Terminology and Nomenclature 3.2 Bi Bi reactions 3.3 Representation of Bi Bi reactions 3.4 Rate equations and double reciprocal plots 4. Differentiating various types of Bi Bi mechanisms 4.2 Product inhibition studies 4.3 Isotope exchange studies 5. Summary

3 1. Learning Outcomes Many enzymes catalyse bisubstrate reactions. Bisubstrate reactions pathways could be different but mechanisms of catalysis all similar and have a nomenclature to classify them. Using kinetic measurements, inhibition kinetics and radioactive exchange studies the different categories of bisubstrate reactions can be identified. 2. Introduction Although the Michaelis-Menten kinetic equation used extensively in studying enzyme kinetics was derived based on single substrate reactions, more than 60% of all enzyme catalysed reactions are bisubstrate in nature. Hence a special study of these would be pertinent. Many oxido-reduction reactions involve a substrate which gets reduced at the expense of a second substrate which gets oxidised resulting in two products. Similarily in the reactions catalysed by the transferases, a functional group from one substrate is transferred to the second substrate again giving rise to two products. The reaction mechanisms in these bisubstrate reactions are different and therefore the present module addresses these mechanisms. 3. Bisubstrate Reactions 3.1 Terminology and Nomenclature Based on a nomenclature introduced by W.W.Cleland for enzyme catalysed reactions, the following terminology is used- 1. Substrates are introduced as A, B, C, D in the order they bind to enzyme. 2. Products are called P,Q, R and S in the order they leave the enzyme. 3. Enzyme forms which occur during the reaction mechanism and are stable are designated E,F,G, H. 4. Uni (one), Bi (two), Ter (three) and Quad (four) are used to describe the number of substrates and products involved in that order. For example a reaction-

4 has two substrates and two products. Hence it is called a Bi Bi reaction. On the other hand a reaction having one substrate and two products is called a Uni Bi reaction. 3.2 Bi Bi Reactions Many group transfer reactions such as catalysed by phosphotransferases (also called kinases) fall into two categories: Sequential or single displacement reactions Here the two substrates A and B must bind first in ordered manner or randomly to the enzyme followed by the release of the products P and Q again in an ordered manner or randomly. Because the two substrates must first bind before the two products are released, hence the terminology- single displacement. Double displacement or Ping Pong reactions. In some Bi Bi reactions a substrate (called leading substrate) first binds to the enzyme followed by the release of the leading product. Then the second substrate is bound following which a second product is released. Because this mechanism resembles a to and fro movement of the ball in a ping-pong ortable tennis game, the mechanism is popularily called Ping-Pong. Two displacements reactions are involved and hence they are also known as double displacement reactions. 3.3 Representation of Bi Bi reactions. Single displacement reactions depending on whether the substrates bind in an orderly or random manner are subclassified into ordered or random single displacement reactions. In double displacement or Ping Pong reactions, the first or leading substrate is A followed by the release of first product P. An ordered Bi Bi reaction a random ordered Bi Bi reaction and a Ping Pong reaction are represented in Fig. 1 a, b, c, respectively. Also to be noted in the representation is that the horizontal line represents the enzyme, substrates meet the line whereas products leave the line. In Ping Pong reactions the enzyme may undergo changes and these are represented as E, F... etc.

5 Fig. 1 Cleland representation of a. ordered Bi Bi reaction, b. random Bi Bi reaction and c. Ping Pong reaction mechanisms. 3.4 Rate equations and double reciprocal plots. For determining the rate of bisubstrate reactions of the type- One of the substrates say B is kept constant and the concentration of A is varied and a plot of 1/Vo vs 1/[A] is made (Lineweaver-Burk plot). In another set of reactions the concentration of A is kept constant and the concentration of B is varied. Again a plot of

6 1/Vo is made vs 1/[B]. Using steady state kinetic measurements and in the absence of product the rate equations given below have been derived for ordered and random Bi Bi reactions. Ordered Bi-Bi Rapid equilibrium random Bi Bi mechanism Fig. 2 Double reciprocal plot of single displacement reactions

7 In both the types of single displacement reactions, the graphs are identical. The lines intercept behind the 1/Vo axis. For a double displacement /Ping Pong type Bi Bi reaction the steady state kinetic equation is given below and the double reciprocal plot is illustrated in Fig. 3. Fig. 3 Double reciprocal plot of Ping Pong Bi Bi mechanism. Here a series of parallel lines are obtained wherein a plot of 1/V o vs 1/[A] at varying concentrations of B indicating identical slopes but intercepts on the 1/V o axis being equal to K M/V max. A series of parallel lines indicates that at constant concentration of B and varying [A] the slope is independent of [B] and the slope is independent of [A] in the plot of 1/V o vs 1/[B]. Thus the pattern of the double reciprocal plots obtained can easily differentiate single and double displacement mechanisms. 4 Differentiating various types of Bi Bi mechanisms In section 3 it was possible to use double reciprocal plots to broadly classify reactions into single and double displacement reaction mechanisms. However using this method, ordered and random single displacement reactions could not be differentiated. In this section few more techniques such as product inhibition and isotope exchange studies are described to arrive at a correct mechanism of the Bi Bi reaction. 4.1 Product inhibition studies

8 In the single displacement reactions two mechanisms occur, ordered and random. In the ordered reaction, the substrates A and B bind in order and the products P and Q leave the enzyme in that order. In such a reaction if excess product P or Q is added the reverse reaction cannot occur as both products are not present together. However binding of P or Q (whichever is added) will inhibit the forward reaction. Consider a mechanism for an ordered reaction wherein a group X is transferred from one substrate to the other resulting in two products- P and Q which is equivalent to B-X (as X is transferred from A to B). If excess Q is added, it will bind to the enzyme preventing the leading substrate A which is equivalent to P-X from binding. In other words, Q would compete with A (competitive inhibitor) when [A] is varied and [B] is fixed. On the other hand Q is a mixed inhibitor of B when [A} is constant, as B binds to EA in the ordered mechanism. However in the random ordered mechanism, which is sometimes known as Rapid Equilibrium Bi Bi mechanism, products P and Q are competitive with respect to [A] and [B]. The following Table 1 summarises these observations. Table 1. Product inhibition patterns in Bi Bi reaction mechanisms 4.2 Isotope exchange studies Although in the previous sections, it is possible to arrive at some conclusion using kinetic data and double reciprocal plots on the mechanism of the Bi Bi reaction occasionally additional experimental data may be useful to come to a definitive conclusion. This is because rates of reactions can be innacurately determined due to the use of one substrate at a constant concentration and varying the other substrate concentration, leading to some confusion. Isotope exchange experiments can corroborate the evidence gathered from kinetic and inhibition studies. Consider two apparently similar reactions catalysed by the enzymes sucrose phosphorylase and maltose phosphorylase. Reaction catalysed by sucrose phosphorylase:

9 Reaction catalysed by maltose phosphorylase: The two above reactions can be represented as: The group which is transferred is X from A to form Q. In a single displacement reaction, X is directly transferred from A to B, the enzyme merely providing a binding site for both substrates to bind and transfer X. On the other hand in a Ping-Pong reaction, the leading substrate A (P-X) binds to E and transfers X to the enzyme transiently converting it to another form F (or E-X) and is released as P or the first product. When the second substrate B binds, this X group is transferred to it and the second product Q (or P-X) is released. Which of the two mechanisms is taking place can be determined by removing one substrate B and instead A is mixed with a small amount of radioactive Q in the presence of enzyme. The reactions occurring would be- If the group X is transferred directly to substrate B then the radioactive product will not exchange as is possible in a single displacement reaction. In the mechanism catalysed by the enzyme sucrose phosphoryllase, if sucrose is mixed with a small amount of radioactive fructose in the absence of phosphate, it is found that the label exchanges and sucrose gets radiolabelled. It is then clear that this can only occur in a Ping-Pong mechanism. In the case of the enzyme maltose phosphorylase, if maltose and radioactive glucose are added to the enzyme, the radiolabel does not exchange, confirming the mechanism to be a single displacement type as indicated in the reactions-

10 5. Summary 60% of all enzyme catalysed reactions are bisubstrate in nature. A special nomenclature and representation of bisubstrate reactions was proposed by W.W.Cleland. Two categories of bisubstrate reaction mechanisms- single displacement and double displacement are found to occur. Single displacement reactions are of two types- ordered and random. These various types of mechanisms can be differentiated and diagnosed using double reciprocal plots, product inhibition studies and isotope exchange studies.