Multiple turnover reactions

Figure 5.9 Multiple turnover reactions that produce chiral amino acids from achiral keto acids.

In the MMO OB3b system, the values could be used to predict the concentration dependence of the MMOB enhancement on the rate of the multiple turnover reaction. The fit to the experimental data predicts that the maximum rate is attained when a stoichiometric ternary complex (based on active site concentration) is established. Excess MMOB is inhibitory, apparently due to the formation of inactive MMOB-MMOR and MMOB-MMOB complexes, or perhaps binding of MMOB in the MMOR binding site. Cross-linking experiments were used to demonstrate the formation of each of these inhibitory complexes. Component complexes also play a significant role during the single turnover reaction as described below. 

Figure 2. Kinetic mechanism of the hairpin ribozyme. Rate constants were derived from pre-steady-state and multiple turnover reactions, using radiolabeled and fluorescent substrates as described (Esteban et al., in press)...

In addition, it has been discovered that there are naturally occurring enzymes that facilitate Diels-Alder type reactions within certain metabolic pathways and that enzymes are also instrumental in forming polyketides, isoprenoids, phenylpropanoids, and alkaloids (de Araujo et al., 2006). Agresti et al. (2005) identified ribozymes from RNA oligo libraries that catalyzed multiple-turnover Diels-Alder cycloaddition reactions. 

A suitable stable transition state analog is hexachloronorbomene derivative 25, which mimics most of the geometrical features of the transition state, including the boat conformation of the cyclohexene ring. It was used to poduce antibodies that catalyse the reaction between 20 and 21 efficiently, with substantial rate acceleration and multiple turnovers. 

Reaction conditions permitting a catalyst to pass through many catalytic rounds. Multiple-turnover conditions are usually obtained by maintaining the substrate concentration in excess over the concentration of active catalyst. This technique usually allows one the opportunity to evaluate the catalytic rate constant ka,t, which is the first-order decay rate constant for the rate-determining step for each cycle of catalysis, and one can evaluate the magnitude of other parameters such as the substrate s dissociation constant or Michaehs constant. 

We specifically chose the reaction between anthracene and maleimide (Figure 5.3.1) for a number of reasons. Most importantly, we assumed that the completely different overall geometry of reactants and products would facilitate enrichment of catalysts that are capable of multiple turnovers. Anthracene is planar, in contrast with the 120° angles between the different rings in the Diels-Alder product. A ligand that can bind to anthracene should, therefore, not be able to bind to the product except after extensive refolding. The availability of sensitive UV absorbance and fluorescence assays for anthracene was another practical reason to choose this reaction. 

Scheme 3. The highly-ordered transition state associated with the Diels-Alder reaction has made it a natural target for work in the field of catalytic antibodies [15]. Indeed, Hilvert [15a] has reported recently the first successfully antibody-catalyzed Diels-Alder reaction. Monoclonal antibodies elicited to the boxed hapten 14 provided both acceleration of and multiple turnover of the cycloaddition between tetrachlorothiopene dioxide and IV-ethyl maleimide. The initial adduct decomposes by the chelotropic-extrusion of sulfur dioxide affording the dihydrophthalimide derivative 13...

Liu L, Zhou W, Chruma JJ, Breslow R. Transamination reactions with multiple turnovers catalyzed by hydrophobic pyridoxamine cofactors in the presence of polyethylenimine polymers. J. Am. Chem. Soc. 2004 126 8136-8137. 

Figure 5 Chemoenzymatic approaches for the production of novel bioactive compounds. In this example, the enzymatic buildup of the linear precursor of daptomycin by its NRPSs (DptA, DptBC, and DptD) is substituted by solid-phase synthesis (a). By using the 4 Ppan transferase Sfp and the CoA-thioester of the linear peptide, the opo-enzyme PCP-TE and be modified, and after trans-esterification cyclized by the TE domain (b). Because the resulting ho/o-enzyme cannot be modified again, this is a single turnover reaction. Another strategy uses thiophenole-esters of the linear peptides to be cyclized (c). When these compounds are used, no PCP domain is necessary. The TE domain is readily acylated, and regiospecific and stereospecific cyclization toward daptomycin or, depending on the linear peptide provided, toward variants thereof occurs. Because the enzyme is not altered in any way after product release, this setup results in a multiple turnover.

In an alternative approach, Nakayama and Schultz [25] have successfully achieved the enantiofacial reduction of prochiral ketones. By utilizing the phosphonate hapten 15 catalytic antibodies were elicited which catalyze a highly stereospecific reduction of ketone 16 with sodium cyanoborohydride as a cofactor (Scheme 4). The most active antibody, A5, was found to have a pH optimum at acidic pH, consequently the reductions were performed in aqueous buffer at pH 5.0. The reaction was followed for multiple turnovers (>25) without any decrease in activity or stereoselectivity highlighting the utility of this catalytic system. 

Although steady-state kinetic methods cannot establish the complete enzyme reaction mechanism, they do provide the basis for designing the more direct experiments to establish the reaction sequence. The magnitude of kcm will establish the time over which a single enzyme turnover must be examined for example, a reaction occurring at 60 sec will complete a single turnover in approximately 70 msec (six half-lives). The term kcJKm allows calculation of the concentration of substrate (or enzyme if in excess over substrate) that is required to saturate the rate of substrate binding relative to the rate of the chemical reaction or product release. In addition, the steady-state kinetic parameters define the properties of the enzyme under multiple turnovers, and one must make sure that the kinetic properties measured in the first turnover mimic the steady-state kinetic parameters. Thus, steady-state and transient-state kinetic methods complement one another and both need to be applied to solve an enzyme reaction pathway. 

Under pre-steady-state conditions, the enzyme in the reaction is used in stoichiometric amounts, meaning that one of the substrates has a concentration smaller than or comparable to the enzyme concentration. In single-turnover DNA polymerase assays of dNTP incorporation, the enzyme concentration is in excess of the DNA substrate concentration. These conditions allow us to follow the enzyme through one complete catalytic cycle, thus eliminating complications from multiple turnovers. Nucleotide incorporation is a relatively fast process occurring on a millisecond timescale, and rapid chemical quench is the specialized instrument generally... 

Studies of Alcohol Oxidation. When the reaction was investigated from the direction of alcohol oxidation under pre-steady-state conditions in the presence of IBA, the time-resolved spectra obtained from RSSF measurements again show evidence for the formation of a transient intermediate in the NAD -mediated oxidation of benzyl alcohol.Data collected at pH values of 9.0, 5.6, and 4.8 are shown in Figs. 7 and 8. In the wavelength region 300 to 450 nm and at pH 9.0, the time-resolved spectra are characterized by a fast, pre-steady-state (exponential) phase dominated by the appearance of bound NADH. This process is followed by an approximately zeroth-order (steady-state) phase in which free NADH is generated by multiple turnovers. The difference spectra in Fig. 7C,D compare the changes which occur in the pre-steady-state phase with those in the steady... 

The vast majority of biochemical reactions are catalyzed by enzymes (Bagg, 2004 Copeland, 2000), which are proteins fulfilling all the three criteria of being biocatalysts efficiency (a), specificity (b) and multiple turnover (c). The term enzymes is reserved for the natural catalytic proteins in this text. Although few quasi-catalytic enzymes are known such as type I restriction endonuclease, poly(ADP-ribose) synthetase and transmethylase for 0 -methylguanine mediate specific reactions with large acceleration but are inactivated in the reaction. 

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