S110: Rate limiting steps for processive hydrolysis by cellobiohydrolase 1

Enzymatic hydrolysis of cellulose exhibits a conspicuous slow-down as the process progresses. This behavior appears ubiquitous for different substrates and enzymes (including enzyme cocktails), and constitutes an essential challenge for cost-effective saccharification of biomass. Thus, in the later stages of a saccharification, the specific enzymatic rate may fall well below 1 catalytic event per minute, and this is about two orders of magnitude less than the initial value.

Many possible causes of the slow-down have been suggested, and these may be coarsely divided into two groups: 1. Restrictions that build up with the degree of conversion and 2. Inherent restrictions governed by enzyme-substrate interactions. Typical examples of factors from group 1 include product inhibition and depletion of reactive subsets of the insoluble substrate. Here, we focus on group 2 and show how the analysis of the initial (transient) kinetics of cellobiohydrolase 1 (CBH1) provides insight into the rates of different steps in the cellulolytic process. Specifically, we have used real-time measurements of cellulase activity on different insoluble substrates and explicit modeling to show that the early slowdown of CBH1 reflects a balance between rapid adsorption, chain acquisition and processive movement on one hand and a slow off-rate on the other.