The Slowdown of the Endoglucanase <i>Trichoderma
reesei</i> Cel5A-Catalyzed Cellulose Hydrolysis Is Related to
Its Initial Activity
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Abstract
One
important
feature of hydrolysis of cellulose by cellulases is that the reaction
slows down quickly after it starts. In this work, we investigate the
slowdown mechanism at the early stage of the reaction using endoglucanase <i>Tr</i>. Cel5A-catalyzed phosphate acid-swollen cellulose
(PASC) hydrolysis as a model system. Specifically, we focus on the
effect of enzyme adsorption on the reaction slowdown. Nineteen single
mutations are introduced (with the assistance of molecular dynamics
simulations) to perturb the enzyme PASC interaction, yielding the
adsorption partitioning coefficient <i>K</i><sub>r</sub> that ranged from 0.12 to 0.39 L/g, compared to that of the wild
type (0.26 L/g). Several residues,
including T18, K26, Y26, H229, and T300, are demonstrated to be important
for adsorption of the enzyme to PASC. The kinetic measurements show
that the slowdown of the hydrolysis is not correlated with the adsorption
quantified by the partitioning coefficient <i>K</i><sub>r</sub> but is anticorrelated with the initial activity. This result
suggests that the mutants with higher activity are more prone to being
trapped or deplete the most reactive substrate faster and the adsorption
plays no apparent role in the reaction slowdown. The initial activity
of Cel5A against PASC is correlated with the enzyme specific activity
against a soluble substrate <i>p</i>-nitrophenyl cellobioside