Enzymes ping-pong reaction

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. 

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 ping-pong reaction occurs, for example, when a phosphate-transferring enzyme, such as phosphoglycerate mutase, is phosphorylated by one substrate to form a phosphorylenzyme (E—P in equation 3.55), which then transfers the... 

In a two-substrate reaction similar to that catalyzed by hexokinase, two basic mechanisms may be at work. First, a ping-pong reaction may be occurring in which the enzyme shuttles between a stable enzyme intermediate, such as a phosphorylated enzyme, and a free enzyme. Second, the reaction may be sequential, in which case no reaction occurs until both substrates are on the enzyme. There are two types of sequential mechanisms. If one substrate cannot bind until after the addition of the other substrate the mechanism is said to be ordered. However, if they can combine in any order the mechanism is said to be random. The various kinetic methods for distinguishing between these mechanistic forms have been summarized by Cleland (52). The evidence for and against these possible kinetic schemes will now be summarized for yeast hexokinase. 

In double-displacement, or Ping-Pong, reactions, one or more products are released before all substrates bind the enzyme. The defining feature of double-displacement reactions is the existence of a substituted enzyme intermediate, in which the enzyme is temporarily modified. Reactions that shuttle amino groups between amino acids and a-keto acids are classic examples of double-displacement mechanisms. The enzyme aspartate aminotransferase (Section 23.3.1) catalyzes the transfer of an amino group from aspartate to a-ketoglutarate. 

The enzyme—substrate interactions can proceed by either single-displacement or double-displacement reactions (commonly known as ping-pong reactions). A substrate reaction proceeding by way of a single-displacement reaction can be shown by... 

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). 

Steady-state kinetics have been used to determine the kinetic mechanisms of many of these enzymes. The questions that have been primarily addressed are the sequence of steps that occur in substrate binding prior and subsequent to the catalytic reaction and the potential formation of covalent enzyme intermediates. Classical interpretation of kinetic analyses has been the determination of the relevant reactions occurring via a random or an ordered sequential reaction, or if the reaction is a double-displacement or Ping-Pong reaction. In the former case, phosphoryl transfer occurs in the ternary complex that contains enzyme, phosphoryl donor, and phosphoryl acceptor. In the latter case, enzyme reacts with... 

In so-called ping-pong reactions products are released in a stepwise fashion. In a two-substrate reaction, the first substrate (S,) binds to the enzyme E and a product (Pi) is released, leaving the enzyme chemically modified (denoted E ), perhaps by a fragment of the substrate. Then the second substrate (S2) binds to the modified enzyme and is processed into a second product, P2, returning the enzyme to its native form. The scheme can be summarized as follows ... 

Lipases are serine hydrolases and follow a bi-bi ping-pong reaction mechanism [2,3]. During the catalysis (Scheme 1) an acyl enzyme is formed in the acylation step where the serine hydroxyl is acylated by the acyl donor (the first substrate, RvCOOR2) [4]. The first product (HOR2) is then released. The acyl acceptor (the second substrate, HOR3), which in the natural reaction is water but can in principle by any nucleophile, then reacts with the acyl enzyme in the deacylation step to form the second product (RjCOORs) and die free enzyme. 

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). 

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. 

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). 

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. 

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.

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