Enzymes ping pong

The catalytic reaction of lipases follow the so called ping-pong bi-bi mechanism, a double displacement mechanism. This is a special multisubstrate reaction in which, for a two-substrate, two-product (i.e., bi-bi) system, an enzyme reacts with one substrate to form a product and a modified enzyme, the latter then reacting with a second substrate to form a second, final product, and regenerating the original enzyme (ping-pong). 

Reactions that fit this model are called ping-pong or double-displacement reactions. Two distinctive features of this mechanism are the obligatory formation of a modified enzyme intermediate, E, and the pattern of parallel lines obtained in double-reciprocal plots (Figure 14.19). 

One class of enzymes that follow a ping-pong-type mechanism are aminotransferases (previously known as transaminases). These enzymes catalyze the transfer of an amino group from an amino acid to an a-keto acid. The products are a new amino acid and the keto acid corresponding to the carbon skeleton of the amino donor ... 

If the enzyme-catalyzed reaction is to be faster than the uncatalyzed case, the acceptor group on the enzyme must be a better attacking group than Y and a better leaving group than X. Note that most enzymes that carry out covalent catalysis have ping-pong kinetic mechanisms. 

In nature, aminotransferases participate in a number of metabolic pathways [4[. They catalyze the transfer of an amino group originating from an amino acid donor to a 2-ketoacid acceptor by a simple mechanism. First, an amino group from the donor is transferred to the cofactor pyridoxal phosphate with formation of a 2-keto add and an enzyme-bound pyridoxamine phosphate intermediate. Second, this intermediate transfers the amino group to the 2-keto add acceptor. The readion is reversible, shows ping-pong kinetics, and has been used industrially in the production ofamino acids [69]. It can be driven in one direction by the appropriate choice of conditions (e.g. substrate concentration). Some of the aminotransferases accept simple amines instead of amino acids as amine donors, and highly enantioselective cases have been reported [70]. 

CO oxidation occurs in two half-reactions (1) oxidation of CO to CO2 generating the two-electron reduced enzyme and (2) reoxidation of the enzyme by the electron acceptor. The ping-pong nature of this reaction was first proposed based on studies with cell extracts from C. thermoaceticum 54) and C. pasteurianum 155). 

Figure 7-4. Ping-pong mechanism for transamination. E—CHO and E—CHjNHj represent the enzyme-pyridoxal phosphate and enzyme-pyridoxamine complexes, respectively. (Ala, alanine Pyr, pyruvate KG, a-ketoglutarate Glu, glutamate).

Figure 8-11. Representations of three classes of Bi-Bi reaction mechanisms. Horizontal lines represent the enzyme. Arrows indicate the addition of substrates and departure of products. Top An ordered Bi-Bi reaction, characteristic of many NAD(P)H-dependent oxidore-ductases. Center A random Bi-Bi reaction, characteristic of many kinases and some dehydrogenases. Bottom A ping-pong reaction, characteristic of aminotransferases and serine proteases.

In ping-pong reactions, one or more products are released from the enzyme before all the substrates have added. 

The emphasis in kinetic studies of E-IIs has been on the analysis of the rates of phosphorylation of the sugar by the phosphoryl group donor. In the early studies the question was addressed whether phosphorylated E-II would be a catalytic intermediate in the reaction or whether the phosphoryl group would be transferred directly from the donor to the sugar on a ternary complex between the enzyme and its substrates [66,75,95-100]. This matter has been satisfactorily resolved by a number of other techniques in favor of the first option and possible reasons why some systems did not behave according to a ping-pong type of mechanism have been discussed [1]. 

The two substrate kinetics of the overall reaction catalyzed by the complex in permeabilized membranes showed classical ping-pong kinetics in accordance with a phosphorylated enzyme intermediate. The affinity constants for fructose and P-enolpyruvate were 8 and 25 /iM, respectively. 

Many studies have demonstrated that enzymes in organic media keep their conventional Michaelis-Menten or ping-pong behavior [10,132,133,137]. Nevertheless, enzyme activity usually decreases and kinetic parameters are dramatically changed when compared to biocatalysis in aqueous media [136]. 

Several mechanisms have been proposed for lipase-catalyzed reactions. Kinetic studies of hydrolysis [14,15] and esterification [50] catalyzed by Pseudomonas cepecia lipase, demonstrate that the enzyme has a ping-pong mechanism. 

Figure 2.15 Double recipcrocal plots for a bi-bi enzyme reactions that conform to (A) a ternary complex mechanism and (B) a double-displacement (ping-pong) mechanism.

The Ping-Pong Mechanism with an Immobilized Enzyme and the Cosubstrate in Solution... 

Figure 8. The most common enzyme mechanisms, represented by their corresponding Cleland plots The order in which substrates and products bind and dissociate from the enzyme is indicated by arrows, (a) The Random Bi Bi Mechanism-. Both substrates bind in random order, (b) The Ordered Sequential Bi Bi Mechanism-. The substrates bind sequentially, (c) The Ping Pong Mechanism-. The enzyme exists in different states E and E. A substrate may transfer a chemical group to the enzyme. Only upon release of the first substrate, the chemical group is transferred to the second substrate.

Certain classes of enzymes tend to have characteristic mechanisms. (Examples transaminases often have ping-pong mechanisms, kinases usually do not). 

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. 

A mathematical equation indicating how the equilibrium constant of an enzyme-catalyzed reaction (or half-reaction in the case of so-called ping pong reaction mechanisms) is related to the various kinetic parameters for the reaction mechanism. In the Briggs-Haldane steady-state treatment of a Uni Uni reaction mechanism, the Haldane relation can be written as follows ... 

A balanced hypothetical chemical equation indicating the transfer of electrons between two different oxidation states of the same element of chemical species. 2. One segment of a ping-pong (or double-displacement) enzyme mechanism. 

PING PONG HALF-REACTIONS. Many enzymes operate by double-displacement mechanisms involving covalent enzyme-substrate intermediates as shown in the following scheme ... 

---