Ping-pong mechanism, enzyme

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).

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. 

Studies of product inhibition357,358 and initial velocity359 of the dehydrogenase reaction are consistent with a hexa uni ping-pong mechanism,359 which requires that an irreversible step of the process should occur in the reaction sequence prior to addition of the second molecule of NAD to the enzyme. The irreversible step is, most probably, formation of the uronic acid (90a) from the aldehyde 93, and several mechanisms may be written to explain the kinetics ob-... 

Although this reaction is fully reversible, the relatively high [ATP]/[ADP] ratio in cells normally drives the reaction to the right, with the net formation of NTPs and dNTPs. The enzyme actually catalyzes a two-step phosphoryl transfer, which is a classic case of a double-displacement (Ping-Pong) mechanism (Fig. 13-12 see also Fig. 6-13b). First, phosphoryl group transfer from ATP to an active-site His residue produces a phosphoenzyme... 

FIGURE 13-12 Ping-Pong mechanism of nucleoside diphosphate kinase. The enzyme binds its first substrate (ATP in our example), and a phosphoryl group is transferred to the side chain of a His residue. ADP departs, and another nucleoside (or deoxynucleoside) diphos-... 

Ping-pong" mechanisms. A common type of mechanism that is especially prevalent for enzymes with tightly bound cofactors has been dubbed ping-pong because the enzyme alternates between... 

One less kinetic parameter can be obtained from an analysis of the data for a ping-pong mechanism than can be obtained for ordered reactions. Nevertheless, in Eq. 9-47, twelve rate constants are indicated. At least this many steps must be considered to describe the behavior of the enzyme. Not all of these constants can be determined from a study of steady-state kinetics, but they may be obtained in other ways. 

A substrate analog will frequently inhibit only one of the two forms of a multisubstrate enzyme with a ping-pong mechanism.1 72 Reciprocal plots made for various inhibitor concentrations consist of a family of parallel lines reminiscent of uncompetitive inhibition. Observation of such parallel line plots can support a ping-pong mechanism for an enzyme but cannot prove it because in some cases parallel lines are observed for inhibition of enzymes acting by an ordered sequential mechanism. The following question arises naturally for any ordered bimolecular reaction (Eq. 9-43) Of the... 

Kinetics. In a double-displacement mechanism the enzyme shuttles between free enzyme and the intermediate carrying the substrate fragment (here, the glycosyl enzyme). With sucrose phosphorylase the maximum velocity varies with the concentrations of sucrose and HP042 in the characteristic fashion expected for this "ping-pong" mechanism (Eq. 9-47)43... 

The following experiments shed some light. Kinetic studies of sucdnyl-CoA-acetoacetate CoA transferase indicate a ping-pong mechanism. The enzyme alternates between two distinct forms, one of which has been shown to contain bound CoA.932-934 The E-CoA intermediate formed from enzyme plus acetoacetyl-CoA was reduced with 3H-containing sodium borohydride and the protein was completely hydrolyzed with HC1. Tritium-containing a-amino-8-hydroxyvaleric acid was isolated. Since thioesters (as well as oxygen esters) are cleaved in a two-step process... 

This process is called the Ping-Pong mechanism to emphasize the bouncing of the enzyme between two states, E and E. Ping-Pong pathways are commonly observed with enzymes that contain bound coenzymes. Interconversion of the enzyme between the two forms usually involves modification of the coenzyme. 

Enzymes often require multiple substrates to complete their catalytic cycle. This may involve combining two compounds into one molecule or transferring atoms or electrons from one substrate to another. The substrates may both bind to an enzyme and react collectively, or each substrate might bind, react, and release sequentially. With two substrates, if both bind to the enzyme, a ternary complex (ES S2) will form (Scheme 4.8). The order of substrate addition may be important (ordered) or not (random order). Cases in which the two substrates react sequentially follow a double-displacement, or ping-pong, mechanism (Scheme 4.9). Enzymes requiring more than two substrates have more complicated complexation pathways. 

LiP catalyzes the oxidation of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) to veratryl aldehyde. Since this reaction can be easily followed at 310 nm, it is the basis for the standard assay for this enzyme (26,27). The enzyme exhibits normal saturation kinetics for both veratryl alcohol and H202 (28,43). Steady-state kinetic results Indicate a ping-pong mechanism in which H202 first oxidizes the enzyme and the oxidized intermediate reacts with veratryl alcohol (43). The enzyme has an extremely low pH optimum ( 2.5) for a peroxidase (43,44) however, the rate of formation of compound I (kx, Fig. 2) exhibits no pH dependence from 3.0-7.0 (45,46). Addition of excess veratryl alcohol at pH 3.0 results in the rapid conversion of... 

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