Substrate Specificity and Inhibition

For the diene there are few structural requirements for acceptance by the 49nt ri-bozyme as a substrate [12]. The diene must contain three linearly annelated rings. Enlargement of the anthracene by introduction of methyl or hydroxymethyl groups at positions 1, 4, 9', and 10 did not impede the action of the ribozyme. Substitutions at the 2- and 3-positions did, however, prevent the ribozyme from accelerating the Diels-Alder reaction. Thus, in a putative binding pocket there seems to be space in front and behind the anthracene but there is tight fit at the sides. The substituent in the 9-position is unnecessary for recognition, because ether, thio-ether, or alkyl residues are accepted; this also indicates no hydrogen bonds are formed between the ribozyme and the anthracene substrate.

For the dienophile some structural requirements are necessary for acceptance by the ribozyme as a substrate (Figure 5.3.7). It must be a five-membered ring and substituents on the reactive double bond are not permitted. The biotinyl residue is not necessary for recognition of maleimide by the 49nt RNA, because the initially used biotin maleimide can be truncated to a maleimide and an alkyl chain, both of

Fig. 5.3.7. Summarized structural requirements of dienes and dienophiles for acceptance by 49nt ribozyme. Substitutions shown in black are tolerated whereas those denoted by empty letters are deleterious. A denotes removal of the whole substituent.

which seem to be essential for catalysis. The activity is maximum for N-pentyl-maleimide. No branching in the a-position is tolerated.

Because of the product-like nature of the transition state of the Diels-Alder reaction, interactions between product and ribozyme might provide valuable mechanistic information. Cycloaddition products and analogs were tested for their ability to inhibit the catalyzed reaction. The requirements for the effect of inhibitors are essentially the same as for substrates. The catalyzed reaction proceeds highly enantioselectively, and product inhibition was also found to be stereospecific. A R,R-product enantiomer inhibits the natural D-ribozyme approximately 20-fold more strongly than the S,S-product. For the mirror image L-ribozyme, the S,S product is the stronger inhibitor. Each ribozyme enantiomer is inhibited more effectively by its respective product. An anthracene with a strongly electron-withdrawing group in the 9-position is no substrate, yet is bound by the catalyst, as demonstrated by its inhibitory effect on the catalyzed reaction.

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