Complex Ping Pong Mechanisms

1 Partial Rapid Equilibrium Ping Pong Bi Bi System 

If the substrate addition and product release steps in the Ping Pong Bi Bi mechanism are much faster than the interconversions of central complexes, then E, EA, and EQ are at equilibrium and also F, FB, and FP are at equilibrium. 

In this case, although the rapid equilibrium segments affect the composition of the kinetic constants in terms of rate constants, the final form of the velocity equation is unchanged. Thus, the reciprocal plots and product inhibition patterns are unchanged. This mechanism will occasionally appear in practice. 

2 Hybrid Theorell-Chance Ping Pong Systems 

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  

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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 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.

Enzymatic catalysis mechanisms may well be considerably more complex than the basic ping-pong mechanism, involving inhibition and hysteresis phenomena. Horseradish peroxidase offers a remarkable example... 

Fig. 2. The Bonnichsen, Chance, and Theorell 34) mechanism for the dismutation of hydrogen peroxide by catalase. (A) The simple ping-pong mechanism (ferric-peroxide compound (ycle) involves only the successive formation and decomposition of the compound 1 intermediate by two successive molecules of H2O2. (B) Reversible ES(Fe -H202) and ternary (compound I-H2O2]) complexes are added to the mechanism in A.

The formation of a ternary complex is entropically disfavoured relative to binary ones. However, kinetic and spectroscopic investigations [39] gave no indication of, e.g., a ping-pong mechanism, and/or the involvement of covalent intermediates... 

MULTISUBSTRATE MECHANISM Random Bi Bi ternary complex mechanism, ISOTOPE EXCHANGE AT EQUILIBRIUM RANDOM Bl UNI MECHANISM RANDOM Bl UNI UNI Bl PING PONG MECHANISM... 

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. 

The kinetics of the yeast lipoamide dehydrogenase in the direction of NAD+ reduction indicate a bi-bi ping-pong mechanism is operative in this species also (117). If the enzyme from yeast indeed proves to have a tighter EHa-NADH complex than does the mammalian enzyme, product inhibitions studies should show impressive dependence on the fixed substrate (9S). 

The discussion here is limited to CuZnSOD for a number of reasons. First, in this system the ping-pong mechanism described in reactions (22) and (23) is operative as written whereas both MnSOD and FeSOD carry out catalysis by mechanisms that involve observable enzyme-substrate complexes under certain conditions. Secondly, this enzymatic system has been studied as a model to look at such factors as electrostatic guidance of substrate (see below). Finally, the link that was demonstrated between over 100 point mutations in CuZnSOD and the inherited version of amyotrophic lateral sclerosis (Lou Gehrig s Disease) has made underscored the importance in understanding details of enzyme function. ... 

If the two-substrate mechanism does not involve the formation of a ternary complex, then sequential substrate binding and product release occurs in the so-called ping-pong mechanism, Eqs. 2.30 and 2.31 ... 

Where there are two substrates, there are two fundamentally different kinetic mechanisms. In the first, the so-called ping pong mechanism, one substrate is bound, is partly transformed at the active site (often with a loss of molecular fragment) and then the second substrate binds and the product is released. In the ternary complex mechanism, by contrast, both substrates have to bind at the active site before any catalysis occurs, after which products are released. The ternary complex mechanism is called sequential in most texts on enzyme kinetics, because the substrates bind in sequence, but here the term will be avoided because the difference from the ping-pong mechanism is not self-evident. 

Mechanism. Dopamine /1-hydroxylase operates via a ping-pong mechanism, in which the oxidized form of the enzyme is reduced by ascorbate (ping). In the second step, dopamine and oxygen bind to the enzyme in a defined sequence creating a tertiary complex. The products are then released following the oxidation of dopamine (pong) [180,181]. 

The chemical and kinetic mechanisms of the eubacterial TGT have been the subject of intense study. Although very few details remain unclear, the main features of the TGT catalytic mechanism are now well established. Kinetically, TGT follows a ping-pong mechanism where tRNA binds first, forming a covalent complex with the enzyme followed by the dissociation of guanine. The heterocyclic substrate (preQj) then binds and finally preQj-tRNA is released. 

The kinetic mechanism of MAO is similar to that of DAAO - it depends on the substrate. In all cases, the amine binds to the enzyme and is oxidized. In some cases, the product dissociates before the enzyme is reoxidized by molecular oxygen, giving a ping-pong mechanism. In other cases, product dissociation is slow and the reduced enzyme-product complex reacts with molecular oxygen, forming the oxidized enzyme-product complex, followed by product dissociation. ... 

The catalytic mechanisms of LOX and LMO are very similar, with one key exception. The rate of pyruvate dissociation is much faster in LOX than in LMO. LOX catalyzes a true ping-pong mechanism, which can be divided into two distinct half-reactions. In the reductive half-reaction, LOX is reduced as lactate is oxidized to pyruvate. ° Flavin reduction occurs at 105 s at 4°C at 25 °C, the reaction is too fast to be measured. A fourfold primary isotope effect is observed on this step when using a- H-lactate as a substrate. The product of lactate oxidation is the reduced enzyme/pyruvate complex, which exhibits long-wavelength charge-transfer absorbance. Product dissociation from this complex occurs at 35 s about 7000-fold faster than pyruvate dissociation in LMO. In the oxidative half-reaction, the free reduced enzyme, formed due to the quick release of pyruvate, is oxidized by molecular oxygen with a second-order rate constant of... 

Fig. 4. Ether phospholipid synthesis from dihydroxyacetone-phosphate. (A) Dihydroxyacetone-P acyl transferase (DHAPAT). The first step of ether phospholipid synthesis is catalyzed by peroxisomal DHAPAT. This enzyme is a required component of complex ether lipid biosynthesis and its role cannot be assumed by a cytosolic enzyme that also forms acyldihydroxyacetone-P. (B) Ether bond formation by alkyl-DHAP synthase. The reaction that forms the 0-alkyl bond is catalyzed by alkyl-DHAP synthase and is thought to proceed via a ping-pong mechanism. Upon binding of acyl-DHAP to the enzyme alkyl-DHAP synthase, the pro-f hydrogen at carbon atom 1 is exchanged by enolization of the ketone, followed by release of the acyl moiety to form an activated enzyme-DHAP complex. The carbon atom at the 1-position of DHAP in the enzyme complex is thought to carry a positive charge that may be stabilized by an essential sulfhydryl group of the enzyme thus, the incoming alkox-ide ion reacts with carbon atom 1 to form the ether bond of alkyl-DHAP. It has been proposed that a nucleophilic cofactor at the active site covalently binds the DHAP portion of the substrate.

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