Product inhibition patterns

This equation reveals atypical product inhibition patterns for a random mechanism P is noncompetitive with both A and B Q is competitive with both A and B. Whenever abnormal product inhibition patterns are ob-... 

A more recent examination of the kinetics of this enzyme by initial rate measurements has included product inhibition patterns and has led to the conclusion that at least under some conditions an ordered bi-bi mechanism applies which involves a ternary complex of enzyme, NAD, and dihydrolipoamide (157). Clear spectral evidence is presented for the existence of a complex between NAD and the oxidized enzyme and this will be discussed in Section III,E. The product inhibition pattern for NAD tended toward that expected for this mechanism only at high NAD concentration. 

The early kinetic studies on glutathione reductase did not include investigation of product inhibition, so vital to a proper interpretation of kinetic data in the elucidation of the mechanism 247, 248). In the one case where product inhibition patterns were observed, they were not interpreted by more recent kinetic theory 40). Subsequent kinetic analyses see below), in which product inhibition patterns have been obtained, were either completed prior to the discovery of the EH2-NADPH complex... 

As pointed out previously in this review the steady-state kinetics of mitochondrial transhydrogenase, earlier interpreted to indicate a ternary Theorell-Chance mechanism on the basis of competitive relationships between NAD and NADH and between NADP and NADPH, and noncompetitive relationships between NAD" and NADP" and between NADH and NADPH, has been reinterpreted in the light of more recent developments in the interpretation of steady-state kinetic data. Thus, although the product inhibition patterns obtained in the earlier reports [75-77] using submitochondrial particles were close to identical to those obtained in a more recent report [90] using purified and reconstituted transhydrogenase, the reinterpretation favors a random mechanism with the two dead-end complexes NAD E NADP and NADH E NADPH. A random mechanism is also supported by the observation that the transhydrogenase binds to immobilized NAD as well as NADP [105] in the absence of the second substrate. 

Thus kcai and are a function of all the rate constants in the pathway and any simplifying assumptions concerning individual rate constants are likely to be inaccurate. Moreover, the three reaction pathways shown in Schemes I and 11, and 111 are indistinguishable by steady-state methods. Although product inhibition patterns provide evidence for the E-P state, individual kinetic constants cannot be resolved. Schemes 11 and 111 reduce to Scheme 1 under the conditions where ki, k2- Steady-state kinetics cannot resolve the three reaction mechanisms because the form of the equation for steady-state kinetics is identical for each mechanism (v = rate) ... 

In the presence of activator, pyruvate, the substrate saturation curves of the R. ruhrum ADP-Glc PPase are hyperbolic at low temperatures. Using kinetic studies its reaction mechanism was studied. The product inhibition patterns eliminated all known sequential mechanisms except the ordered BiBi or Theorell—Chance mechanisms. Small intercept effects suggested the existence of significant concentrations of central transis-tory complexes. Kinetic constants obtained in the study also favored the ordered BiBi mechanism. In addition studies using ATP-[ P]-pyrophosphate isotope exchange at equilibrium supported a sequential-ordered mechanism, which indicated that ATP is the first substrate to bind and that ADP-Glc is the last product to... 

The compulsory ordered mechanism arrived at through isotopic exchange rates is basically in agreement with mechanisms postulated on the basis of initial velocity studies. Heyde and Ainsworth showed that for beef heart m-MDH the initial velocity pattern in the absence of products and the product inhibition pattern are consistent with an ordered mechanism (77). Raval and Wolfe obtained similar results with pig heart m-MDH data obtained by initial velocity studies in both directions are in agreement with a compulsory ordered mechanism (78,79). Substrate inhibition by oxaloacetate also occurs with pig heart m-MDH (80). Similar initial velocity studies on beef heart s-MDH did... 

The malate activation of m-MDH has been explained in terms of malate binding at some effector site, a site other than the active site (89). It is suggested that the malate activation can be accounted for primarily by a tighter binding of NAD+ (89). These data and product inhibition patterns are in general agreement with a compulsory ordered mechanism described in an earlier section. 

TABLE 11.5 Cleland nomenclature for bisubstrate reactions exemplified. Three common kinetic mechanisms for bisubstrate enzymatic reactions are exemplified. The forward rate equations for the order bi bi and ping pong bi hi are derived according to the steady-state assumption, whereas that of the random bi bi is based on the quasi-equilibrium assumption. These rate equations are first order in both A and B, and their double reciprocal plots (1A versus 1/A or 1/B) are linear. They are convergent for the order bi bi and random bi bi but parallel for the ping pong bi bi due to the absence of the constant term (KiaKb) in the denominator. These three kinetic mechanisms can be further differentiated by their product inhibition patterns (Cleland, 1963b)... 

Table 2- Product inhibition patterns in rapid equilibrium bisubstrate mechanisms Plowman, 1972 Segel, iQ75)... 

However, the primary double reciprocal plots of some rapid equilibrium systems are identical. In rapid equilibrium systems, in the presence of the products of reaction, the primary reciprocal plots are very characteristic and depend on the number and type of enzyme-substrate and enzyme-product complexes that can form. Therefore, in order to distinguish between different t5q>es, one must revert to product inhibition patterns that can easily distinguish between aU types of rapid equilibrium bisubstrate systems (Plowman, 1972 Segel, 1975) (Table 2). 

Therefore, initial velocity studies alone will not distinguish between the two mechanisms however, the missing ABP and BPQ denominator terms lead to different product inhibition patterns. 

The product inhibition patterns in the Theorell-Chance mechanism are different from those of the Ordered Bi Bi mechanism. Note that the product inhibition equations are symmetrical Equations (9.34) and (9.38) are symmetrical and so are Eqs. (9.35) and (9.37). Thus, product inhibition studies only identify A-Q and B-P pairs and do not reveal the order of substrate addition and product release. 

Isomerization of a stable enzyme form does not affect the algebraic form of the rate equation in the absence of products, but product inhibition patterns are modified so that the order of addition of substrates and release of products can not be determined by steady-state kinetic experiments. Rate constants for steps involving the isomerizing stable form or any central complex are not determinable, and steady-state distributions can be calculated only for non-isomerizing... 

Some hyperbolic bisubstrate mechanisms can be easily distinguished by their primary double reciprocal plots in the absence of products, such as ordered from the Ping Pong mechanism. However, in many cases, the bisubstrate mechanisms cannot be distinguished in this way. Fortunately, in most cases, they can be clearly separated on the basis of their product inhibition patterns (Plowman, 1972) (Table 3). 

Table 3. Product inhibition patterns in bisubstrate mechanisms (Plowman, 1972 Fromm, 1975,1979)... 

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. 

In the absence of products, aU initial velocity patterns are parallel, in the same way as in the Ping Pong Bi Bi mechanism. However, the product inhibition patterns are different, and may serve to distinguish between the different systems (Table 6). 

Let us illustrate the product inhibition patterns with an example of the Ordered Ter Ter mechanism (Section 12.4). In the presence of all the substrates of reaction, A, B, C, and the product Q, the rate equation is... 

Therefore, in order to simplify the product analysis of trisubstrate reactions, and for a proper interpretation of product inhibition patterns, we shall need a suitable comparative overview of trisubstrate mechanisms. Table 5 lists the product inhibition patterns for the major Ter Bi and Ter Ter mechanisms. Table 5 shows that, ultimately, each mechanism can be identified unequivocally on the basis of its unique product inhibition patterns. Product inhibition analysis is also able to identify unequivocally each substrate in a given mechanism (Plowman, 1972 Fromm, 1975, 1995). 

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