Single Turnover Reaction Sequence

In each of these studies the siterproduct relationship was determined on the basis of product distribution data obtained from standard, steady state catalytic reactions. While this approach can provide evidence for the type of site(s) responsible for the formation of certain products it cannot give any indication of the number of such sites that are present on the catalyst surface. Since the activities of the various types of sites are different, it is possible that a small number of very active sites could dominate product formation. In order to relate the extent of produet formation with the number of specific types of sites present an experimental arrangement is needed which obviates these site activity differences. One way of doing this is to use the catalyst surface as a stoichiometric reagent so that each site reacts only once. In this way there will always be a 1 1 site product molecule ratio regardless of the rates at which the different types of sites react. 

This has been accomplished with the single turnover (STO) procedure shown in Fig. 3.9.51,64-66 this reaction sequence, a sample of the catalyst is 

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Fig. 3.9. Single turnover reaction sequence. (Reproduced, with permission, from Ref. 66).

We have used our Single Turnover (STO) reaction sequence to characterize dispersed metal catalysts with respect to the numbers of alkene saturation sites, double bond isomerization sites, and hydrogenation inactive sites they have present on their surfaces (ref. 13). Comparison of the product composition observed when a series of STO characterized Pt catalysts were used for cyclohexane dehydrogenation with those observed using a number of instrumentally characterized Pt single crystal catalysts has shown that the STO saturation sites are comer atoms of one type or another on the metal surface (ref. 10). 

For combination in a twin ribozyme both units should specifically interact with a unique substrate sequence. To this end we changed the substrate sequence in the single units and determined the kinetic constants of the cleavage reaction before constructing the twin ribozymes. All single catalytic modules under single turnover conditions had rate constants between 0.2 and 0.5 min-1. 

Hill, B. C., 1994, Modeling the sequence of electron transfer reactions in the single turnover of reduced, mammalian cytochrome c oxidase with oxygen, J. Biol. Chem. 269 2419n2425. 

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. 

The Schiff base (27) formed in the initial phase of the reaction catalyzed by lumazine synthase can be observed in single-turnover experiments as an optical transient with an absorption maximum at 330nm. A later optical transient with an absorption maximum at 445 nm has been assigned to the product resulting from phosphate elimination. Surprisingly, the ring closure reaction at the end of the reaction sequence appears as the rate-determining step. ... 

A complete kinetic scheme has been established for the enzyme from both sources. The L. casei dihydrofolate reductase followed a reaction sequence identical to the E. coli enzyme (Scheme I) moreover, none of the rate constants varied by more than 40-fold Figure 20 is a reaction coordinate diagram comparing the steady-state turnover pathway for E. coli and L. casei dihydrofolate reductase, drawn at an arbitrary saturating concentration (1 mM) of NADPH at pH 7. The two main differences are (i) L. casei dihydrofolate reductase binds NADPH more tightly in both binary (E-NH, -2 kcal/mol) and tertiary (E NH-H2F, - 1.4 kcal/mol E-NH-H4F, - 1.8 kcal/mol) complexes, and (ii) the internal equilibrium constant (E-NH H2F E-N-H4F) for hydride transfer is less favorable for the L. casei enzyme (1 kcal/mol). These changes, as noted later, are smaller than those observed for single amino acid substitutions at the active site of either enzyme. Thus, the overall kinetic sequence as well as the... 

After five cycles of selection and ampHfication, a population of single-stranded DNAs was enriched that catalyzed the Pb +-dependent cleavage at the ribose residue. This intramolecular cleavage activity was transformed into an inter-molecular reaction by separating the 38-nucleotide long catalytic domain from the 21-mer substrate which was cleaved specifically and with high turnover rates. Remarkably, the deoxyribozyme can perform well only with the special DNA/RNA chimeric oHgonucleotide substrate and cannot cleave a pure RNA substrate of the same sequence. 

A catalytic cycle is defined by a closed sequence of elementary steps, i.e., a sequence in which the active site is regenerated so that a cyclic reaction pattern is repeated and a large number of turnovers occurs on a single active site [5]. If the stoichiometric equation for each of the steps in the cycle is multiplied by its stoichiometry number, i.e., the number of times it occurs in the catalytic cycle, and this sequence of steps, the reaction pathway, is then added, the stoichiometric equation for the overall reaction is obtained. This equation must contain only reactants and products because all intermediate species must cancel out, and this overall reaction is represented by an equal sign ... 

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