Ping-Pong Bisubstrate Reactions

A ping-pong mechanism is one in which a molecule of one product is released between the addition of the two reactants.24 The equilibria for this type of reaction is shown in Eqn. 7.62. 

In this sequence the metal site, M, adsorbs a substituent, A, to give the M-A complex. This is changed to another complex, M -P, which dissociates to give one of the products, P, and a modified site, M. M then reacts with the second substrate, B, to give M -B which converts to M-Q then desorbs the second product, Q, and regenerates the initial active site, M. 

The reciprocal form of the rate expressions for this reaction sequence run, with [A] changing and [B] constant, is shown in Eqn. 7.63. The reverse gives an equation symmetrical in A and B. 

The plots of 1/v versus 1/(A] at several concentrations of B are shown in Fig. 7.14. These plots are characterized by a series of parallel lines having a slope of Km(a/V max The y-axis intercepts are given by Eqn. 7.64 and the x-axis intercepts by Eqn. 7.65. 

The replot of the y-axis intercepts versus 1/(AJ has a slope of I M(B) max y-axis intercept of Wmax x-axis intercept of -1/K vi(b). Replotting the x-axis intercepts from Fig. 7.14 versus 1/(81 gives a line with a slope of a y-axis intercept of 1/K vi(yv) x-axis intercept of 

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The proposed mechanism for the catalytic cycle of ID-1 involves a ping-pong bisubstrate reaction in which the selenol form of the enzyme (E-SeH) first reacts with T4 to form a selenenyl iodide intermediate (E-Sel) with release of the deiodin-ated compound T3. The subsequent reaction between the selenenyl iodide intermediate and an unidentified thiol cofactor regenerates the selenol form. 

In contrast to class I flavin reductases, class II flavin reductases are bona fide flavoproteins producing a characteristic flavin-absorption spectrum and containing a bound flavin prosthetic group involved in the electron transfer from NAD(P)H to the flavin substrate. The reaction sequence for class II enzymes, as exemplified by the flavoprotein component of sulfite reductase from E. coli, follows a ping pong bisubstrate-biproduct mechanism (85). In this process, the NAD(P)H and flavin bind to the same site sequentially electrons are first transferred from NADPH to FAD and then from FAD to another flavin. 

Almost all enzymes—in contrast to the simplified description given on p. 92—have more than one substrate or product. On the other hand, it is rare for more than two substrates to be bound simultaneously. In bisubstrate reactions of the type A + B C+D, a number of reaction sequences are possible. In addition to the sequential mechanisms (see p.90), in which all substrates are bound in a specific sequence before the product is released, there are also mechanisms in which the first substrate A is bound and immediately cleaved. A part of this substrate remains bound to the enzyme, and is then transferred to the second substrate B after the first product C has been released. This is known as the ping-pong mechanism, and it is used by transaminases, for example (see p.l78). In the Lineweaver— Burk plot (right see p.92), it can be recognized in the parallel shifting of the lines when [B] is varied. 

Km and Umax have different meanings for different enzymes. The limiting rate of an enzyme-catalyzed reaction at saturation is described by the constant kcat, the turnover number. The ratio kcat/Km provides a good measure of catalytic efficiency. The Michaelis-Menten equation is also applicable to bisubstrate reactions, which occur by ternary-complex or Ping-Pong (double-displacement) pathways. 

There are three general patterns observed with bisubstrate reactions random, sequential and ping-pong. 

In some bisubstrate reactions, no ternary complex ES1S2 is formed, because the binding of the first substrate is followed by release of the first product before the second substrate is bound and the second product is released. This sequence is described as a ping-pong bi-bi type of reaction. It occurs in reactions catalyzed by aminotransferases. 

Figure. 6.10. Ping-pong mechanism for bisubstrate reactions.

TABLE 11.5 Cleland nomenclature for bisubstrate reactions exemplified. Three common kinetic mechanisms for bisubstrate enzymatic reactions are exemplified. The forward rate equations for the order bi bi and ping pong bi hi are derived according to the steady-state assumption, whereas that of the random bi bi is based on the quasi-equilibrium assumption. These rate equations are first order in both A and B, and their double reciprocal plots (1A versus 1/A or 1/B) are linear. They are convergent for the order bi bi and random bi bi but parallel for the ping pong bi bi due to the absence of the constant term (KiaKb) in the denominator. These three kinetic mechanisms can be further differentiated by their product inhibition patterns (Cleland, 1963b)... 

There are two possible bisubstrate systems that combine the enzyme feature of the Ping Pong sequence with the hit-and-mn feature of the Theorell-Chance mechanism. These are in fact the hmiting cases of the common Ping Pong Bi Bi system, in which one of two central complexes has extremely short life. The reaction sequences are shown below ... 

Bisubstrate reactions (Chapters 8 and 9). In bisubstrate reactions, a frequent case is a need to distinguish between the Steady-State Ordered, Ping Pong, and Equihbrium Ordered mechanism the rate equations involved are... 

Lipase-catalyzed reactions do not necessarily require water as the second reactant. An immobilized enzyme is often stable in anhydrous media (Sharma et al 2001) and can use different alcohols as the secondary acceptors of fatty acids. The concentration of alcohol is relatively low and cannot be treated as a constant. Consequently, the reaction obeys the bisubstrate ping-pong mechanism (Cheirsilp et al 2008 Mitchell et al., 2008 Xiong et al., 2008) as shown in Figure 14.3. The corresponding equation of the reaction rate in the absence of products is ... 

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