Dead-end complexes

The origin of the remarkable stereoselectivities displayed by chiral homogeneous catalysts has occasioned much interest and speculation. It has been generally assumed, using a lock-and-key concept, that the major product enantiomer arose from a rigid preferred initial binding of the prochiral olefin with the chiral catalyst. Halpren 48) on the basis of considerable evidence, reached the opposite conclusion the predominant product enantiomer arises from the minor, less stable diastereomer of the olefin-catalyst adduct, which frequently does not accumulate in sufficient concentration to be detected. The predominant adduct is in essence a dead-end complex for it hydrogenates at a much slower rate than does the minor adduct. 

Nonproductive reversible complexes of an enzyme with various substrates and/or products. The International Union of Biochemistry distinguishes dead-end complex from abortive complex, and the latter term is regarded... 

The reduction in enzymatic activity that results from the formation of nonproductive enzyme complexes at high substrate concentration. The most straightforward explanation for substrate inhibition is that a second set of lower affinity binding sites exists for a substrate, and occupancy of these sites ties up the enzyme in nonproductive or catalytically inefficient forms. Other explanations include (a) the removal of an essential active site metal ion or other cofactor from the enzyme by high concentrations of substrate, (b) an excess of unchelated substrate (such as ATP" , relative to the metal ion-substrate complex (such as CaATP or MgATP ) which is the true substrate and (c) the binding of a second molecule of substrate at a subsite of the normally occupied substrate binding pocket, such that neither substrate molecule can attain the catalytically active conformation". For multisubstrate enzymes, nonproductive dead-end complexes can also result in substrate inhibition in the presence of one of the reaction... 

CIRCADIAN RHYTHM dCMP DEAMINASE Dead-end complexes,... 

In addition, a noncompetitive mechanism has also been observed in some peptides, and this means that the peptide can combine with an enzyme molecule to produce a dead-end complex, regardless of whether a substrate molecule is bound or not. For example, LIY (Nakagomi et al.,... 

DDT dehydrochlorinase 551 Dead-end complexes 466 DEAE-cellulose 104 DEAE-Sephadex 106 Deamidation... 

These compounds act as alkylating agents N2 is released and a nucleophilic group from the enzyme becomes attached at the carbon atom indicated.134 Other inhibitors bind noncovalently to form dead-end complexes.1343... 

Competitive inhibitors often closely resemble in some respect the substrate whose reactions they inhibit and, because of this structural similarity, compete for the same binding site on the enzyme. The enzyme-inhibitor complex either lacks the appropriate reactive groups or is held in an unsuitable position with respect to the catalytic site of the enzyme which results in a complex which does not react (i.e. gives a dead-end complex). The inhibitor must first dissociate before the true substrate may enter the enzyme and the reaction can take place. An example is malonate, which is a competitive inhibitor of the reaction catalysed by succinate dehydrogenase. Malonate has two carboxyl groups, like the substrate, and can fill the substrate binding site on the enzyme. The subsequent reaction, however, requires that the molecule be reduced with the formation of a double bond. If malonate is the substrate, this cannot be achieved without the loss of one of the carboxy-groups and therefore no reaction occurs. 

ESI is the dead-end complex the inhibitor constant K, = ES -J— Under steady state conditions ES ... 

Irreversible inhibitors are those that permanently disable the enzyme. The complex El or EIS cannot dissociate, so that these are dead-end complexes. When an irreversible inhibitor is added to the enzyme-containing solution, inhibition may not be complete immediately, but increases gradually with time, as more and more enzyme molecules are modified. 

Establishing the inhibition patterns in an enzyme-catalyzed reaction is usually an important step in elucidating the reaction mechanism. One complication in the interpretation of such data is the possible formation of dead-end complexes (i.e., a complex of the form EAP in the above scheme). This is especially important in rapid-equilibrium reactions [ones in which all steps except the rate constants for the central isomerization step (EAB EPQ in the above example) are very large]. 

Kinetic studies of the Fe(SOD) indicate a fairly simple oxidation-reduction cycle in which 02 is bound to the Fe(III) form of the protein and oxidized to 02, followed by binding of a second 02 to the resulting Fe(II) form and reduction to H202. The kinetics of Mn(SOD) are more complicated and the turnover number (1300 s-1 at 25 °C) is much lower than for Fe(SOD) (26,000 s-1) however, a similar catalytic cycle is believed to occur. The manganese enzyme kinetics are complicated by a side reaction to form a dead end complex, possibly a Mn(III) peroxide complex (61, 62). 

Cleland (160), steady-state kinetics of a Theorell-Chance mechanism can generally apply also to a rapid-equilibrium random mechanism with two dead-end complexes. However, in view of the data obtained with site-specific inhibitors this latter mechanism is unlikely in the case of the transhydrogenase (70, 71). The proposed mechanism is also consistent with the observation of Fisher and Kaplan (118) that the breakage of the C-H bonds of the reduced nicotinamide nucleotides is not a rate-limiting step in the mitochondrial transhydrogenase reaction. 

The means by which NAD affects the oxidation of NADH is still uncertain. The evidence for two pyridine nucleotide binding sites is not compelling. The alternative explanation that NAD functions by reversing the equilibrium between EHj and 4-electron-reduced enzyme (EH4) is shown in Eq. (9). There is some kinetic evidence for a dead end complex... 

The reactions in Eq. (10), proceeding to the right from E, are part of normal catalysis as shown in Fig. 10 (SI, S3). The association of E with NADP leads to a dead end complex. In the reaction of yeast glutathione reductase with NADPH, EH,-NADPH appears to be formed in the dead time of the rapid reaction spectrophotometer (co. 3 msec) when observation is at 540 nm (344), however, if 3.4 /iM EH,(free) is mixed with 20 fiM NADPH, Eq. (11), a minimum rate of complex formation of... 

The enzyme-bound inhibitor may either lack an appropriate functional group for further reaction, or may be bound in the wrong position with respect to the catalytic residues or to other substrates. In any event, the enzyme-inhibitor complex E. I is unreactive (it is sometimes referred to as a dead-end complex) and the inhibitor must dissociate and substrate bind before reaction can take place. 

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. 

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 2 7, 2 8). 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 53) or have not considered it. Furthermore, the product inhibition patterns have been carried out at only a single level of the fixed substrate it is essential that the patterns be obtained at more than one level of fixed substrate, especially where dead end complexes are involved 249) as has been so amply demonstrated with lipoamide dehydrogenase 95, 157). In spite of these deficiencies, the more recent kinetic studies have yielded much useful information. 

In the reverse reaction, driving the synthesis of S3P and PEP from EPSP and phosphate, the amplitude of the product formation was altered because of the formation of a dead-end complex with S3P and phosphate bound to the active site. The time dependence of this inhibition during the single turnover to form E-S3P would lead to a model too complex to solve explicitly. The reaction time... 

The pig heart enzyme is very fast in the physiological direction (NAD to NADH) with a turnover number of 550 s at pH 7.6 and 25 °C. In the opposite direction (NADH to NAD ), NAD has an activating effect because excess NADH will over-reduce the enzyme to EH4, which is not competent for catalysis (Scheme 16). NAD" " oxidizes this dead-end complex, increasing the amount of EH2. The enzyme from E. coli is especially susceptible to inhibition from over-reduction by NADH because the redox potentials of EH2 and EH4 are closer than in enzymes from other sources. As a consequence of closer potentials, the EH2 state... 

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