Product inhibition patterns mechanisms

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)... 

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. 

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). 

Product inhibition studies are used to complement kinetic analyses and to distinguish between ordered and random Bi-Bi reactions. For example, in a random-order Bi-Bi reaction, each product will be a competitive inhibitor regardless of which substrate is designated the variable substrate. However, for a sequential mechanism (Figure 8-11, bottom), only product Q will give the pattern indicative of competitive inhibition when A is the variable substrate, while only product P will produce this pattern with B as the variable substrate. The... 

Fromm and Rudolph have discussed the practical limitations on interpreting product inhibition experiments. The table below illustrates the distinctive kinetic patterns observed with bisubstrate enzymes in the absence or presence of abortive complex formation. It should also be noted that the random mechanisms in this table (and in similar tables in other texts) are usually for rapid equilibrium random mechanism schemes. Steady-state random mechanisms will contain squared terms in the product concentrations in the overall rate expression. The presence of these terms would predict nonhnearity in product inhibition studies. This nonlin-earity might not be obvious under standard initial rate protocols, but products that would be competitive in rapid equilibrium systems might appear to be noncompetitive in steady-state random schemes , depending on the relative magnitude of those squared terms. See Abortive Complex... 

In practice, uncompetitive and mixed inhibition are observed only for enzymes with two or more substrates—say, Sj and S2—and are very important in the experimental analysis of such enzymes. If an inhibitor binds to the site normally occupied by it may act as a competitive inhibitor in experiments in which [SJ is varied. If an inhibitor binds to the site normally occupied by S2, it may act as a mixed or uncompetitive inhibitor of Si. The actual inhibition patterns observed depend on whether the and S2-binding events are ordered or random, and thus the order in which substrates bind and products leave the active site can be determined. Use of one of the reaction products as an inhibitor is often particularly informative. If only one of two reaction products is present, no reverse reaction can take place. However, a product generally binds to some part of the active site, thus serving as an inhibitor. Enzymologists can use elaborate kinetic studies involving different combinations and amounts of products and inhibitors to develop a detailed picture of the mechanism of a bisubstrate reaction. 

The determination of kinetic mechanisms requires more than just initial velocity patterns, and inhibition studies are usually required. Several types of inhibitors are useful. The products are substrates in the reverse reaction and thus have some affinity for the enzyme and will give inhibition unless their inhibition constants exceed their solubility. Dead-end inhibitors are molecules that play musical chairs with the substrates for open portions of the active site but do not react. Substrates may act as dead-end inhibitors by combining at points in the mechanism where they are not intended and thus cause substrate inhibition. The inhibition patterns caused by these inhibitors are useful in distinguishing between different kinetic mechanisms. 

Three mies can be formulated to predict product or dead-end inhibition patterns in such mechanisms. (1) When an inhibitor occupies the same portion of an active site as the variable substrate, the inhibition is competitive. (2) When an inhibitor combines in a different portion of the same active site as the variable substrate, the inhibition is noncompetitive. Thus, with pyruvate carboxylase, MgADP and P are both noncompetitive inhibitors versus bicarbonate, although in accordance with Rule 1 they are competitive versus MgATP. (3) When an... 

K = 9 pmoll cat/- m = 2.5 X 10 mol Ms ) of PNSHH, resulting in a very high enantiopreference for the (i )-enantiomer. The conversion of (ii )-PNSHH follows an ordered Uni—Bi mechanism, and the inhibition pattern of bromide ion as well as the occurrence of burst kinetics suggested that the bromide ion is first released from the enzyme, followed by the epoxide product para-nmostyrene oxide, PNSO). In addition, multiple turnover analyses showed that the binding of (f( )-PNSHH occurs in a rapid equilibrium step and that the rate of... 

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