Transient Kinetics and Rate-Limiting Steps for the Processive Cellobiohydrolase Cel7A: Effects of Substrate Structure and Carbohydrate Binding Domain

Abstract

Cellobiohydrolases are exoacting, processive enzymes that effectively hydrolyze crystalline cellulose. They have attracted considerable interest because of their role in both natural carbon cycling and industrial enzyme cocktails used for the deconstruction of cellulosic biomass, but many mechanistic and regulatory aspects of their heterogeneous catalysis remain poorly understood. Here, we address this by applying a deterministic model to real-time kinetic data with high temporal resolution. We used two variants of the cellobiohydrolase Cel7A from Hypocrea jecorina , and three types of cellulose as substrate. Analysis of the pre-steady-state regime allowed delineation rate constants for both fast and slow steps in the enzymatic cycle and assessment of how these constants influenced the rate of hydrolysis at quasi-steady state. Processive movement on the cellulose strand advanced with characteristic times of 0.15-0.7 s per step at 25 °C, and the rate was highest on amorphous substrate. The cellulose binding module was found to raise this rate on crystalline, but not on amorphous, substrate. The rapid processive movement signified high intrinsic reactivity, but this parameter had marginal influence on the steady-state rate. This was because dissociation and association were slower and, hence, rate limiting. Specifically, the dissociation from the strand was found to occur with characteristic times of 45-100 s. This meant that dissociation was the bottleneck, except at very low substrate loads (0.5-1 g/L), where association became slower.

Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

“…More recently, Knott et al (55) have used molecular dynamics simulation and free energy calculation to suggest that the rate-limiting step in the hydrolytic chain of reactions is glycosylation of the nucleophile (Glu-212), which had a rate constant of about 11 s Ϫ1 . This theoretical result is in remarkable accordance with k cat values derived from either high speed atomic force microscopy (60) or pre-steady-state kinetic measurements (24). Knott et al (55) also used computational methods to analyze the kinetics of other steps, including processive motion, deglycosylation, catalytic activation, and "threading" the cellulose strand into the active tunnel, but these latter steps were found to be faster than glycosylation, which was therefore suggested to be potentially rate-limiting.…”

“…This latter assumption has previously proven valid for TrCel7A hydrolyzing both Avicel (24) and other substrates (19,22). If we insert the processivities for WT intact (n ϭ 23) and W38A intact (n ϭ 16) found in Fig.…”

“…As outlined in the Introduction, this focus was motivated partly by recent discussions of rate limitation, which have highlighted these two steps, and partly by the deduction that the hydrolytic step (governed by k cat in Scheme 1) is an unlikely candidate for rate determination. The latter relies on numerous measurements of k cat for TrCel7A, which have shown that at room temperature this parameter is in the range of 2-10 s Ϫ1 (19,21,22,24,43,55,60,61), and this is much faster than typically measured specific rates. Because the overall rate is obviously dictated by the slowest step, this discrepancy strongly suggests that catalysis is not the bottleneck and that rate determination must be some much slower process.…”

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

“…the experimentally measured processivity on a real polymer) is much lower than P Intr predicted from the k cat /k off ratio, indicating that the true processive ability is seldom realized. Furthermore, numerous studies have pointed out that the overall rate of processive cellulose hydrolysis is limited by the slow dissociation of the cellobiohydrolases (12,17,(21)(22)(23)(24)(25)(26). This raises the question as to why processive cellulases have evolved to such low k off values.…”

“…In case the TrCel7A-generated insoluble reducing groups on reduced cellulose under single-hit conditions were fluorescence-labeled, the k off value on the order of 10 Ϫ3 s Ϫ1 was obtained (17). Using the global kinetic modeling of progress curves (12,26) and single molecule fluorescence imaging (28), k off values for TrCel7A in the order of 10 Ϫ2 s Ϫ1 were obtained. Using high speed atomic force microscopy, the k off values measured for TrCel7A were in the order of 10 Ϫ1 s Ϫ1 (29,30).…”