Substrate-template ternary complex

Clearly, product dissociation by this scheme, step 3, will limit turnover, as the product-template duplex will be stabilized over the substrate-template ternary complex. Maximizing AAG in step 1 is required to enhance fidelity, but this increased affinity is generally present in the product duplex. Obviously if the binding affinity could be a tuned variable, high substrate affinity for ligation to maximize accuracy and reduced product affinity to enhance catalytic efficiency, product inhibition could be avoided. 

Templates possessing two hydrogen bonding subunits bind two substrates forming a ternary complex in which the substrates are positioned so as to facilitate bond formation between them [5.64a]. In a related way, the rate and stereoselectivity of a bimolecular Diels-Alder reaction are substantially increased by binding both the diene and the dienophile within the cavity of a tris-porphyrin macrocycle [5.64b]. 

T.R. Kelly et al. have synthesized bisubstrate reaction templates utilizing the Chichibabin amination reaction during the preparation of one precursor. This reaction template was designed to use hydrogen bonding to bind two substrates simultaneously but transiently, giving rise to a ternary complex, which positions the substrates in an orientation that facilitates their reaction. 

Scheme 1-12 The template 44 simultaneously binds two substrates (45 and 46) in a ternary complex, accelerating reaction between them.

Scheme 1-15 Self-replicating system designed by Rebek and co-workers. The template 57 (X = OCgF5) binds to the two substrates 55 and 56, generating a ternary complex 55-57-S6. Then 55 and 56 react to generate another molecule of template 57. Stability constants are shown for the 55-56 and 57-57 complexes.

The reaction is facilitated by a linear template T. We will assume that this template has two independent, identical, binding sites both bind substrates with the same microscopic binding constant K, to give a binary complex S-T and a ternary complex S T S. 

RatCTemp (d), f af untemp ( ) d Rate (f) are plotted as functions of [S]q in Figure 1-5 (assuming /fej = 10 s and 2 = 1 s m ). The rate of the reaction in the absence of template (e) increases very steeply with concentration, because it is bimolecular, whereas the rate of reaction via S T S (f) levels off where [S]q > l/K, when all of the substrate is bound, and is asymptotic with a limiting value of [S]ofei/2. There is a certain value of [S]o at which the reaction in the absence of template (e) becomes greater than the maximum possible rate of reaction in the ternary complex (i.e., when / 2[S]o > i/2). When [S]o = kil(2k2), the presence of the template simply doubles the overall rate of reaction (d) and as [S]q increases above this value, the effect of the template becomes insignificant. The critical concentration ki/k2 is a key quantity for defining the effectiveness of a template and it is known as the effective molarity of the system, EM [52]. 

Crystal structures of the NS5B polymerase alone and in complexes with nucleotide substrates have been solved and applied to discovery programs (Ago et al. 1999 Bressanelli et al. 2002 Bressanelli et al. 1999 Lesburg et al. 1999 O Farrell et al. 2003). From these studies, HCV polymerase reveals a three-dimensional structure that resembles aright hand with characteristic fingers, palm, and thumb domain, similar to the architectures of the RNA polymerases of other viruses. However, none of these experimental structures contained the ternary initiation complex with nu-cleotide/primer/template, as obtained with HIV RT. Accordingly, HCV initiation models have been built using data from other viral systems in efforts to explain SAR (Kozlov et al. 2006 Yan et al. 2007). 

The recent determination of the crystal structure of a ternary catalytic complex of HW-1 RT with a substrate (dTTP) and the DNA-primer and template [121] (Fig. 8) has provided the structured basis of resistance it has been found that most mutations causing resistemce to nucleoside-analog drugs are located closely to the nucleoside binding site. 

The construction of catalysts for bimolecular reactions represents a special challenge. Due to entropic reasons, the product- catalyst complex is likely to be more stable than the ternary substrate-catalyst-complex. Consequently, turnover is often low or not even observed. For Diels-Alder reactions, the difficulty to obtain turnover is further increased by the fact that the transition-state and the final product are similar in shape. Nevertheless, a catalytic MIP for a Diels-Alder reaction has successfully been prepared [17]. The trick employed to overcome the problem of similarity between TSA and product is the utilization of a reaction in which the product spontaneously decomposes (Fig. 9). The same reaction had been previously studied with catalytic antibodies. For the catalytic MIP, significant rate enhancements and Michaelis-Menten kinetics were observed. Addition of the template reduces the rate of the reaction to 41% of the original value whereas the control... 

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