A general correction to initial rates determined for nonprocessive exo-depolymerases acting on both substrate and product

Authors: STOLLER, J. ROSE - Oak Ridge Institute For Science And Education (ORISE); Wagschal, Kurt; Lee, Charles; Jordan, Douglas

Submitted to: Analytical Biochemistry
Publication Type: Research Notes
Publication Acceptance Date: 2/9/2017
Publication Date: 4/15/2017
Citation: Stoller, J.R., Wagschal, K.C., Lee, C.C., Jordan, D.B. 2017. A general correction to initial rates determined for nonprocessive exo-depolymerases acting on both substrate and product. Analytical Biochemistry. 523:46-49. doi: 10.1016/j.ab.2017.02.004.
DOI: https://doi.org/10.1016/j.ab.2017.02.004

Interpretive Summary: One of our project goals is to identify highly active enzymes that could perform well in saccharification reactors to help convert hemicelluloses to constituent monomers, which can then be fermented to bioethanol and other valuable products. In working on an exo-polygalacturonase¸ which has a low Km, we found that the enzyme consumed a substantial portion of the first product in the reactions. In order to calculate an accurate initial rate for the first substrate we needed to account for the enzyme acting on the product. We do so in this paper. The example presented is general for exo-polymerases.

Technical Abstract: We recently reported on the kinetics of the polygalacturonase TtGH28 acting on trimer and dimer substrates. When the starting substrate for hydrolysis is the trimer, the product dimer is also subject to hydrolysis, resulting in discrepancies when either the concentration of dimer or monomer product is used for analysis of trimer hydrolysis. Here, we derive a method for determining catalytic rates of exo-hydrolases acting on trimer (and higher order) substrates when products may also be substrates for hydrolysis and show how this correction may be applied for TtGH28.