DNAzyme product-release limitation

DNAzyme product-release limitation is a kinetic regime in which slow departure of cleaved products delays reuse of a DNAzyme, even when the chemical cleavage step is fast. Product formation and regeneration of free catalyst are separate events. A DNAzyme turnover experiment must therefore distinguish one cleavage per catalyst from repeated cycles.

Consider an intentionally simplified saturated-substrate cycle,Here $ES$ is an enzyme-substrate complex and $EP$ a catalyst–product complex. If the two irreversible steps have exponential waiting times and binding of fresh substrate is effectively immediate, the mean DNAzyme catalytic-cycle waiting time is the sum of their means:With stipulated $k_{\mathrm{chem}}=1{\mathrm{s}}^{-1}$ and $k_{\mathrm{rel}}=0.01{\mathrm{s}}^{-1}$, the average cycle takes 101 s and $k_{\mathrm{turn}}\approx 0.00990{\mathrm{s}}^{-1}$. Making chemistry ten times faster reduces the cycle only to 100.1 s. The slow release dominates this model; the numbers are not measurements of a particular DNAzyme.

Under those assumptions a rapid first cleavage can precede much slower continued production. A burst alone is not a unique identification of slow release: catalyst inactivation or mixed active populations can also change the trace. Comparing chemical cleavage with independent product-dissociation observations is more informative than assigning a mechanism from curve shape alone.

The regime is not a definition of every DNAzyme. The primary kinetic analysis by S. W. Santoro and Gerald F. Joyce instead reported substrate-binding limitation of catalytic efficiency for their RNA-cleaving DNA enzyme under the studied conditions.^[ref] Different concentrations and measured quantities can expose different limiting steps; a claim about efficiency at low substrate is not automatically a claim about the slowest saturated turnover step.