Transient kinetic analysis for studying ionizations in RNA modification enzyme mechanisms.

ABSTRACT

The application of in vitro kinetic tools has the potential to provide important insight into the molecular mechanisms of RNA modification enzymes. Utilizing quantitative biochemical approaches can reveal information about enzyme preferences for specific substrates that are relevant for understanding modification reactions in their biological contexts. Moreover, kinetic tools have been powerfully applied to identify and characterize roles for specific amino acid residues in catalysis, which can be essential information for understanding the molecular basis for human disease, as well as for targeting these enzymes for potential therapeutic interventions. RNA methyltransferases are a particularly interesting group of RNA modification enzymes because of the diversity in structure and mechanism that has been revealed among members of this group, even including some examples of enzymes that use entirely distinct reaction mechanisms to form identical methylated nucleotides in RNA. Yet, many questions remain unanswered about how these distinct catalytic strategies are facilitated by the relevant enzyme families. We have applied in vitro kinetic analysis to specifically focus on catalytically relevant ionizations in the context of tRNA methyltransferase reactions, by measuring rates under conditions of varied pH. This analysis can be applied broadly to RNA methyltransferases to expand our understanding of these important enzymes.