“…. k 2 and the first regime holds true (Fig+ 5, triangles)+ If cleavage is not affected by the chase, k Ϫ1 , , k 2 and the second regime fits (Fig+ 5, squares)+ Certain hammerheads have a rate of substrate dissociation on the order of the rate of chemistry, k Ϫ1 Х k 2 so that cleavage is seen during the chase period (Fig+ 5, diamonds)+ In this relatively unusual case, k Ϫ1 can be determined directly from the chase experiment by one of two methods (Fedor & Uhlenbeck, 1992)+ For some hammerhead sequences, the kinetic regime can be changed by altering the pH of the reaction and thereby changing k 2 (Werner & Uhlenbeck, 1995;Clouet-d'Orval & Uhlenbeck, 1996)+ Because k Ϫ1 is not expected to depend on pH, changing the pH effectively alters the relative values of the two rate constants and potentially creates regime-one conditions+ Determining the kinetic regime for a hammerhead by the pulse-chase experiment defines the strategy to obtain k 1 and k Ϫ1 , the elemental rate constants for the binding step+ For hammerheads in the first regime, k 1 and k Ϫ1 cannot be determined directly, although K d ϭ k Ϫ1 /k 1 is easily obtained (see next section)+ For hammerheads in the second regime, k 1 can be obtained by measuring the rate of cleavage at subsaturating ribozyme concentration where binding is rate-limiting+ Under these conditions, k obs ϭ k 1 [R]+ The value of k 1 has only been measured for a limited number of hammerheads, but a value between 10 7 and 10 8 M Ϫ1 min Ϫ1 is usually obtained (Fedor & Uhlenbeck, 1992;Hertel et al+, 1994)+ This value is similar to k 1 values reported for simple RNA duplexes in high concentrations of monovalent cation (Pörschke & Eigen, 1971;Pörschke et al+, 1973;Nelson & Tinoco, 1982)+ However, k 1 values of DNA helices in buffers containing MgCl 2 are often faster (Williams et al+, 1989), suggesting that faster hammerhead association rates may be possible+…”