The hammerhead ribozyme catalyzes a transesterification of a 3',5'-phosphodiester bond, through the attack of the 2'-hydroxyl of a specific nucleotide within the core on its adjacent phosphodiester to produce 2',3'-cyclic phosphodiester and a free 5'-hydroxyl termini in a Mg dependent reaction(Doudna,1994). The reaction leads to the recycle of the ribozyme and the substrate strand cleaved into two.

Figure 1. Schematic of Cleavage Reaction Mechanism (Adapted from Birikh, 1996)

       Mg²⁺ plays a crucial role in the catalysis of hammerhead ribozymes. As shown in Fig.1 above, the C nucleotide is involved in aromatic stacking associations with bases in the uridine-turn. This organization plays an important part in Mg²⁺  binding. This configuration places the 2'-hydroxyl of C in the area of the suggested Mg²⁺ binding site. First, the magnesium complex binds the pro-R oxygen at the phosphate-cleavage site. As the magnesium ion then deprotonates the 2'-hydroxyl of C, nucleophillic attack of the 2'-alkoxide on the scissile phosphate occurs, producing a pentacoordinate phosphate intermediate. This yields the 2',3'-cyclic phosphate and 5'-hydroxyl products. When Mg²⁺ binds to the hydroxyl group, it acts as a basic cofactor as well as supports correct folding of the RNA. While Mg²⁺ is required, other divalent ions such as Mn²⁺, Ca²⁺, Co²⁺, and to a much lesser degree, Sr²⁺ and Ba²⁺ can act as cofactors.

   The removal of 2'-OH group will eliminate the catalysis of the ribozyme. This property was applied to the X-ray crystallography, in which the substrate strand is replaced by DNA so that a stable E-S hybrid can be achieved (Pley, 1994). 

    Like protein-based enzymes, ribozyme catalysis can be described in terms of the Michaelis-Menten mechanism, provided certain assumptions are made. The reaction is composed of three steps: "binding of the ribozyme to the target; cleavage of the target; and release of the cleavage products (Rossi, 1997)

                 Figure 2. The kinetics of the hammerhead ribozyme catalysis. E represents the ribozyme and P1, P2 are two product strands.

    The assumptions are:

1.[ribozyme] < [substrate]

2. E + S <=> ES is rapid and reversible

3. The conversion of ES to EP₁P₂ is the rate determining step.

   K_m and k_cat, were analyzed for the hammerhead ribozyme. K_m is k₁/k_-1 and is the affinity for a particular substrate (usually in the range of 50-500nM) and k_cat is usually about 1 min^-1 (Fedor and Uhlenbeck, 1992). These parameters vary depending upon whether the ribozymes or substrates fold into anomalous, inactive conformations or the conditions of the experiment, such as pH, Mg²⁺ concentration, etc.