Fig. 5.3.4. Mutation analysis of the tripartite ribozyme version (absolutely conserved nucleotides are in black and underlined). The activity profile is shown on a logarithmic scale.

between RNA and substrate was omitted [10]. By employing UV-visible spectroscopy the progress of the reaction could be observed by following the decrease of the anthracene absorption at 365 nm. The catalytic action of this ribozyme on different anthracene and maleimide derivatives was explored and turned out to be successful. Thus, this Diels-Alderase ribozyme is among the first that catalyze chemical reaction between two small organic molecules and remains unchanged after multiple catalytic cycles.

The kinetics of the ribozyme-catalyzed reactions were determined by measuring the initial rates of the catalyzed reaction. For reasons of solubility, anthracene-hexaethyleneglycol, and maleimidocaproic acid were used for these measurements. The initial velocities were determined by monitoring the decrease of anthracene absorbance at 365 nm over @5% conversion. The initial rate of the ribozyme-catalyzed reaction was found to be proportional to the ribozyme concentration. The reaction shows saturation-type kinetics with respect to both substrates. Catalysis was examined as a completely random bireactant system.

The secondary double-reciprocal plot of the data from Figure 5.3.5 gives Michaelis constants of 0.37 mM for the diene and 8 mM for the dienophile. The calculated maximum rate Vmax is 0.15 mM min-1, which at a ribozyme concentration of 7 mm corresponds to a kcat of 21 min-1. The highest initial rate that was measured directly corresponds to a catalytic turnover of approximately 6 min-1. With these catalytic properties the 49nt Diels-Alderase ribozyme is among the faster

5.3.7 Kinetics

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