Determination of Catalytic Parameters for Hairpin Ribozymes

Abstract

Ribozymes have been successfully designed to downregulate gene expression in vivo. To enhance the probability of success in vivo, the investigator should have available the ribozyme with the highest catalytic activity feasible. This can only be determined by in vitro assays for catalytic efficiency and engineering the ribozyme appropriately to optimize this catalytic efficiency. Catalytic efficiency is k _cat/k _m , where k _cat is the turnover number of the reaction and k _m the true Michaelis constant. The k _cat and K _m values are best obtained individually by measuring catalytic activity in reactions where ribozyme is limiting and a range of excess substrate concentrations are used such that the ribozyme turns over (1). Functionally, k _m is the substrate concentration required to achieve half-maximum reaction velocity (V _max), and the turnover number k _cat is obtained by dividing the V _max by the ribozyme concentration (V _max/[Rz]) since V _max = k _cat[Rz]. The k _m is a combination of the individual rate constants comprising the overall reaction and, as such, does not reflect the rate of any given reaction step.