| Abstract: | Stacking between aromatic amino acids and nucleic acid bases may play an important role in the interaction of enzymes with nucleic acid substrates. In such circumstances, disruption of base aromaticity would be expected to decrease enzyme activity on the modified substrates. We have examined the requirement for DNA base aromaticity of five enzymes that act on single-stranded DNA, T4 polynucleotide kinase, nucleases P1 and S1, and snake venom and calf spleen phosphodiesterases, by comparing their kinetics of reaction with a series of dinucleoside monophosphates containing thymidine or a ring-saturated derivative. The modified substrates contained either cis-5R,6S-di-hydro-5,6-dihydroxythymidine (thymidine glycol) or a mixture of the 5R and 5S isomers of 5,6-dihydrothymidine. It was observed that for all the enzymes, except snake venom phosphodiesterase, the parent molecules were better substrates than the dihydrothymidine derivatives, while the thymidine glycol compounds were significantly poorer substrates. Snake venom phosphodiesterase acted on the unmodified and dihydrothymidine molecules at almost the same rate. These results imply that for all the remaining enzymes base aromaticity is a factor in enzyme-substrate interaction, but that additional factors must contribute to the poorer substrate capacity of the thymidine glycol compounds. The influence of the stereochemistry of the dihydrothymidine derivatives was also investigated. We observed that nuclease P1 and S1 hydrolysed the molecules containing 5R-dihydrothymidine approximately 50-times faster than those containing the S-isomer. The other enzymes displayed no measurable stereospecificity. |
