Kinetic parameters of Rnt1p cleavage of inter- and intra-molecular substrates

The K'M and kcat values were determined by measuring the initial rate of production of the 34 and 33 nt cleavage products of 3′-Branch and EL3′-11:TL respectively, as a function of substrate (or complex) concentration. The calculations were performed using the equation one site binding (hyperbola) from Prism 4.0 (GraphPad) and the Michaelis-Menten equations. Errors in the values of the K'd are within±0.10 µM. The indicated values represent the average of three independent measurements using 5′-end labeled substrates. The maximum kcat error limits are±0.07 min−1, the KM error limits are±0.05 µM and the kcat/K'M error limits are±0.09 L•min−1•µM−1.