Are cellulases slow? Kinetic and thermodynamic limitations for enzymatic breakdown of cellulose

Abstract

• The thermodynamic driving force for cellulose hydrolysis is very small • Cellulases hydrolyze glucosidic bonds at a rate comparable to other glucoside hydrolases • Cellulase slowness is generally linked to tight substrate binding and low substrate accessibility • Cellulases show a distinctive negative correlation of substrate binding strength and catalytic turnover • Introduction of a parameter enumerating attack sites on the cellulose surface enables comparative biochemical analyses of cellulases • The apparent specificity constant for cellulases is high compared to glucoside hydrolases acting on soluble substrate. Cellulases are of paramount interest for upcoming biorefineries that utilize residue from agriculture and forestry to produce sustainable fuels and chemicals. Specifically, cellulases are used for the conversion of recalcitrant plant biomass to fermentable sugars in a so-called saccharification process. The vast literature on enzymatic saccharification frequently refers to low catalytic rates of cellulases as a main bottleneck for industrial implementation, but such statements are rarely supported by kinetic or thermodynamic considerations. In this perspective, we first discuss activation barriers and equilibrium conditions for the hydrolysis of cellulose and how these parameters influence enzymatic turnover. Next, we propose a simple framework for kinetic description of cellulolytic enzyme reactions and show how this can pave the way for comparative biochemical analyses of cellulases acting on their native, insoluble substrate. This latter analysis emphasizes that cellulases are characterized by extraordinarily low off-rate constants, while other kinetic parameters including specificity constants and rate constants for association and bond cleavage are quite like parameters reported for related enzymes acting on soluble substrates.

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