Substrate binding active ternary complex

Substrates may affect enzyme kinetics either by activation or by inhibition. Substrate activation may be observed if the enzyme has two (or more) binding sites, and substrate binding at one site enhances the alfinity of the substrate for the other site(s). The result is a highly active ternary complex, consisting of the enzyme and two substrate molecules, which subsequently dissociates to generate the product. Substrate inhibition may occur in a similar way, except that the ternary complex is nonreactive. We consider first, by means of an example, inhibition by a single substrate, and second, inhibition by multiple substrates. 

Perhaps the most important general conclusions from isotope exchange studies, supported by other investigations, are that the preferred pathway mechanism is common to a number of dehydrogenases and that for most, but not all, the formation of the active ternary complex with the second substrate increases the firmness of coenzyme binding, presumably by a protein conformational change. 

Kinetic studies of reversible inhibition by substrate analogs give evidence of the mode of action of the inhibitor and the types of enzyme-inhibitor complex formed, and estimates of their dissociation constants. The complexes may be isolated and sometimes crystallized. Studies of the stabilities, optical properties, and structures of ternary complexes of enzymes, coenzymes, and substrate analog in particular, as stable models of the catalytically active ternary complexes or of the transition state for hydride transfer (61,79,109,115-117), can only be touched upon here there is direct evidence with several enzymes that the binding of coenzymes is firmer in such complexes than in their binary complexes (85,93,118), which supports the indirect, kinetic evidence already mentioned for a similar stabilization in active ternary complexes. 

Information extracted from kinetic data collected under burst conditions, in which there is a two- to fourfold excess of DNA substrate over DNA polymerase, illustrates another important application of presteady-state experiments. This type of experiment provides useful information about the transient concentration of kinetically active ternary complex. A time course of DNA product formation under these conditions demonstrates a transient exponential phase followed by a steady-state linear phase. By examining the dependence of the burst amplitude on DNA concentration, the enzyme s binding affinity for DNA can be evaluated. 

The catalytic activities of Rubisco require an activation process, during which a lysine residue reacts with an activator CO2 molecule (2), which is not the substrate CO2. The labile carbamate formed in this reaction is stabilized by binding of a magnesium ion. The substrate ribulose-1,5-bisphosphate binds to the activated ternary complex and is subsequently either carboxylated by CO2 or... 

Partial protection by bicarbonate may indicate that the carbamylated enzyme is more resistant to photoinactivation. Although non-substrate bicarbonate does bind to the active site, and actually competitively inhibits RuBP binding (6), anions such as formate, and perhaps acetate would be expected to behave similarly. The lack of protection by these compounds (5) suggests that the mode of bicarbonate protection may be due to carbamylation. The lack of any effect by Mg+ suggests that it is the binding of CO2 (or bicarbonate) that confers resistance to photomodification, and not the formation of the active ternary complex enzyme C02 Mg+. ... 

When cobalt is substituted for zinc at the active site, a similar complex is formed.1396 The absorption spectra also show a large red shift for the substrate maximum upon binding to form the ternary complex. Binding also causes a small shift in the d-d band. 

Imidazole also acts as a substrate-competitive inhibitor, forming both binary complexes with LADH, and ternary complexes in the presence of coenzyme. X-Ray studies show that imidazole also binds to the. catalytic zinc by displacing the water molecule.1361 The presence of imidazole at the active site also enhances the rate of carboxymethylation14658 of Cys-46 with both iodoacetate and iodoacetamide.1420 This enhancement of alkylation has become known as the promotion effect .1421 Imidazole promotion also improves the specificity of the alkylation.1422 Since Cys-46 is thought to be alkylated as a metal-thiol complex, imidazole, on binding the active site metal, could enhance the reactivity by donating a electrons to the metal atom, which distributes the increased electron density further to the other ligands in the coordination sphere. The increased nucleophilicity of the sulfur results in promoted alkylation.1409... 

In single displacement reactions both substrates A and B simultaneously must be present on the active site of the enzyme to yield a ternary complex EAB in order that the reaction may proceed. Single displacement reactions take place in two forms, random and ordered, and they are distinguished by the way the two substrates bind to the enzyme. 

Scheme 11.1 also summarizes other impressive examples of the performance of the CBS method [1-8]. Several excellent reviews on the CBS method have appeared recently [1, 2], and no detailed discussion of the development of the process or substrate scope shall be presented in this review. Please note, however, that the oxazaborolidine-catalyzed borane reduction of ketones is a prime example of bi-functional catalysis [2, 9] - as shown in Scheme 11.2, the current mechanistic picture involves simultaneous binding of both the ketone and the borane to the Lewis-acidic (boron) and Lewis-basic (nitrogen) sites of the catalyst A. In the resulting ternary complex B, the reaction partners are synergistically activated toward hydride transfer. 

Actually, it is noteworthy that there are examples of heme-containing dioxygenases that bind 02 with formation of a heme—Fe(II)—02 complex. This is the case of indolamine-2,3-dioxygenase (EC 1.13.11.11) and tryptophan-2,3-dioxygenase, which catalyze the insertion of 02 into L-tryptophan to yield A-for-mylkynurenine [21], The catalytic cycle involves the ternary complex L-trypto-phan-iron(II) enzyme-02 as an active intermediate. In this ternary complex, which yields A-formylkynurenine and the Fe(II) enzyme, 02 and/or the substrate is activated [21], a situation clearly different from that found for PGHS. 

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