Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose

Abstract

The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 μM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178-1186. © 2015 Wiley Periodicals, Inc.

The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics

“…Reference source not found.. Product inhibition can be assumed to be negligible in these experiments because the maximum sugar concentrations measured here (~15 uM) are much less than reported inhibition constants for Cel7A on insoluble substrates [10][11][12][13][14][15] .…”

Exo‐exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo‐lytic character of the enzymes

“…The transition between the fast-and slow phase occurs at 5-10 mM cellobiose, which corresponds to the conversion of less than 0.1% of the CMC, and we deduce that this small population of CMC is readily available as substrate for the CBHs, possibly through exo-attack. Another possible reason for the transition in Figure 5A is product inhibition, but firstly this would not be expected to show a discrete change as in the figure and secondly the inhibition constant for cellobiose of Cel7A acting on polymeric substrate is hundredths of mM (Gruno et al, 2004;Olsen et al, 2016;Teugjas and V€ aljam€ ae, 2013), and product inhibition would therefore only induce insignificant effects on the overall rates in Figure 5A. 5B) and estimates based on the degree of substitution of the CMC.…”

“…These concentrations compare well to the location of the transition in Figure 5A, and we conclude that the degree of substitution of the CMC does not contradict the above interpretation of the transition point. Another possible reason for the transition in Figure 5A is product inhibition, but firstly this would not be expected to show a discrete change as in the figure and secondly the inhibition constant for cellobiose of Cel7A acting on polymeric substrate is hundredths of mM (Gruno et al, 2004;Olsen et al, 2016;Teugjas and V€ aljam€ ae, 2013), and product inhibition would therefore only induce insignificant effects on the overall rates in Figure 5A. The inhibition of Cel6A by cellobiose is still lower (Murphy et al, 2013;Teugjas and V€ aljam€ ae, 2013), and it appears that product inhibition is an unlikely cause for the sharp change of trace in Figure 5A.…”

pH profiles of cellulases depend on the substrate and architecture of the binding region

“…Inspection of the activity data in Figures 1 and S2 allowed us to assess possible interference from product (cellobiose) inhibition. If we consider TrCel7A at room temperature, the final cellobiose concentration at the optimal pH was around 0.07 mM and this is 1-2 orders of magnitude lower than typically reported inhibition constants, which fall in the 0.5-15 mM range depending on the experimental approach and substrate (Gruno, Väljamäe, Pettersson, & Johansson, 2004;Murphy et al, 2013;Olsen et al, 2016;Teugjas & Väljamäe, 2013). Product inhibition of Cel7A decreases rapidly at higher temperatures (Teugjas & Väljamäe, 2013), and it is hence likely also to be negligible above room temperature.…”