Ternary complex mechanisms

A critical feature of the random ternary complex mechanism is that for either substrate the dissociation constant from the binary enzyme complex may be different from that of the ternary enzyme complex. For example, the Ks value for AX dissociation from the E AX complex will have a value of K v<. The affinity of AX for the enzyme may, however, be modulated by the presence of the other substrate B, so that the dissociation constant for AX from the ternary E.AX.B complex may now be c/Xax, where a is a constant that defines the degree of positive or negative regulation of the affinity of AX for the enzyme by the other substrate. The overall steady state velocity equation for this type of mechanism is given by Equation (2.15) ... 

For either of the ternary complex mechanisms described above, titration of one substrate at several fixed concentrations of the second substrate yields a pattern of intersecting lines when presented as a double reciprocal plot. Hence, without knowing the mechanism from prior studies, one can not distinguish between the two ternary complex mechanisms presented here on the basis of substrate titrations alone. In contrast, the data for a double-displacement reaction yields a series of parallel lines in the double reciprocal plot (Figure 2.15). Hence it is often easy to distinguish a double-displacement mechanism from a ternary complex mechanism in this way. Also it is often possible to run the first half of the reaction in the absence of the second substrate. Formation of the first product is then evidence in favor of a doubledisplacement mechanism (however, some caution must be exercised here, because other mechanistic explanations for such data can be invoked see Segel, 1975, for more information). For some double-displacement mechanisms the intermediate E-X complex is sufficiently stable to be isolated and identified by chemical and/or mass spectroscopic methods. In these favorable cases the identification of such a covalent E-X intermediate is verification of the reaction 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.

For bisubstrate reactions that conform to a ternary complex mechanism (see Chapter 3), inactivation should require the presence of the noncognate substrate. 

Fig. 6. A set of possible relations between volume changes in a two-substrate reaction with a random ternary-complex mechanism. Note that these volume changes as defined by Eq. (77) are dissociation volumes.

Random Bi Bi Ternary Complex Mechanism The random or noncompulsory ordered mechanism is noticeably symmetrical, with two different paths for producing the EAB complex from free enzyme E and its substrates A and B, as well as two different pathways for producing the EPQ complex from free enzyme E and its products P and Q ... 

These qualitative conclusions can be justified in terms of rate equations derived for each exchange catalyzed by ordered and random ternary complex mechanisms . ... 

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

The mechanism of enzyme catalysis drawn, using (a)random ternary complex theory, (b)ordered ternary complex mechanism and (c) ping-pong bi-bi mechanism ... 

Ternary-complex mechanism refers to order bi bi mechanism. 

Figure 4.9 Basic compulsory-order ternary-complex mechanism. The basic ordered mechanism for the general reaction A + B P + Q, with a = [A], b = [B], p = [P], and q = [Q] is illustrated. The four states are unbound enzyme (E), enzyme-substrate A complex (E-A), enzyme-substrate A-substrate B complex (E-AB), and enzyme-product Q complex (E-Q).

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. 

In the case of enzymes working via a ternary complex mechanism, we have two extreme cases. The easiest to comprehend is the rapid equilibrium random mechanism (Scheme 5.4) this is the mechanism where the chemistry is most likely to be rate determining and kinetic isotope effects or structure-reactivity correlations are likely to be mechanistically informative. Enzymes acting on their physiological substrates at optimal pH are likely to show a degree of preference for one or the other substrate binding first, but they can often be induced to revert to a rapid equilibrium random mechanism by the use of non-optimal substrates or pH. 

The other extreme form of ternary complex mechanism occurs when the second substrate will only bind after the first substrate has bound - for example, the binding of the first substrate may induce a conformation change which opens up the active site for the binding of the second substrate. Under such circumstances, rapid equilibrium will be observed only for very poor substrates. The kinetic scheme for a system in which glycosyl donor binds first, as is common for glycosyl transferases, is given in Scheme 5.5 and the rate law in eqn. (5.19). 

Scheme 5.5 Ordered equilibrium ternary complex mechanism.

Scheme 8 Two mechanistic proposals for the catalytic mechanism of CoA-transferases. In mechanism A, an acyl-enzyme Intermediate Is formed by reaction of an enzyme-bound glutamate (aspartate for Class III enzymes) with the donor acyl-CoA, followed by the formation of an enzyme-bound glutamyl- (or aspartyl-) CoA thioester Intermediate. The thioester subsequently reacts with the acceptor carboxylate to give a new acyl-enzyme anhydride from which the acyl group Is transferred to CoA. In Class I transferases, this process follows classical ping-pong kinetics, whereas In Class III enzymes the donor carboxylate only leaves the enzyme complex upon formation of the product (see text for details). Mechanism B represents a ternary complex mechanism as used by Class II enzymes In which a transient anhydride made up of the donor and acceptor acyl groups Is formed by reaction of the acceptor carboxylate with the donor acyl-ACP. The free ACP subsequently reacts with this anhydride to complete acyl transfer.

One mechanism which may apply to a two-substfate reaction is the so-called random ternary-complex mechanism. In this mechanism the enzyme E can form binary complexes EA and EB with the two substrates A and B. It can also form the ternary complex EAB, with no restriction on the order in which A and B are attached. In Figure 10.4 this mechanism is represented in two different ways. The lower one is a shorthand notation introduced by the American biophysical chemist... 

The random ternary-complex mechanism two alternative representations. 

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