Single-turnover cleavage, kinetics

For combination in a twin ribozyme both units should specifically interact with a unique substrate sequence. To this end we changed the substrate sequence in the single units and determined the kinetic constants of the cleavage reaction before constructing the twin ribozymes. All single catalytic modules under single turnover conditions had rate constants between 0.2 and 0.5 min-1. 

Schiff base, or quinoidal intermediates do not appreciably affect the rate of the a reaction.90113 (2) L-serine and amino acids such as O-methyl-L-serine that form ES III do stimulate the a reaction.113 (3) The rate of indole-3-glycerol phosphate turnover is roughly correlated with the rate of formation of ES III for each of the amino acids.90-113 (4) The kinetics of the lag in cleavage of indole-3-glycerol phosphate and synthesis of L-tryptophan under single turnover conditions correspond to the rate of ES III formation... 

The model for a—j3 intersubunit communication indicates that it is the formation of the aminoacrylate species that leads to activation of the a reaction. When both serine and IGP are added simultaneously to the enzyme in a single enzyme turnover experiment, there is a lag in the cleavage of IGP that is a function of the reaction of serine to form the aminoacrylate species. Accordingly, amino acids other than serine that can undergo dehydration to form the aminoacrylate such as cysteine should serve as alternate substrates but should lead to a longer lag for the a subunit activation as determined by transient kinetic analysis. Cysteine does... 

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