Background:The molecular understanding of factors that limit enzymatic hydrolysis of cellulose remains incomplete. Results: Pre-steady-state analysis of cellulolytic activity provides rate constants for basic steps of the overall reaction. Conclusion: Slow dissociation of inactive enzyme-cellulose complexes governs the hydrolytic rate at pseudo-steady state. Significance: Kinetic constants elucidate molecular mechanisms and structure-function relationships for cellulases.

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Background:To elucidate the rate-determining steps of cellobiohydrolase Cel7A from T. reesei, variants with lower substrate affinity were designed. Results: Mutant (W38A) had reduced substrate affinity but a 2-fold increase in the maximum quasi-steady-state rate. Conclusion: Dissociation of stalled TrCel7A is the rate-limiting step in the initial phase of hydrolysis. Significance: This work offers a new perspective for the design of faster cellulases.

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.

Serotonin (5-hydroxytryptamine, 5-HT) is a prevalent neurotransmitter throughout the animal kingdom. It exerts its effect through the specific binding to the serotonin receptor, but recent research has suggested that neural transmission may also be affected by its nonspecific interactions with the lipid matrix of the synaptic membrane. However, membrane-5-HT interactions remain controversial and superficially investigated. Fundamental knowledge of this interaction appears vital in discussions of putative roles of 5-HT, and we have addressed this by thermodynamic measurements and molecular dynamics (MD) simulations. 5-HT was found to interact strongly with lipid bilayers (partitioning coefficient ~1200 in mole fraction units), and this is highly unusual for a hydrophilic solute like 5-HT which has a bulk, oil-water partitioning coefficient well below unity. It follows that membrane affinity must rely on specific interactions, and the MD simulations identified the salt-bridge between the primary amine of 5-HT and the lipid phosphate group as the most important interaction. This interaction anchored cationic 5-HT in the membrane interface with the aromatic ring system pointing inward and a prevailing residence between the phosphate and the carbonyl groups of the lipid. The unprotonated form of 5-HT shows the opposite orientation, with the primary amine pointing toward the membrane core. Partitioning of 5-HT was found to decrease lipid chain order. These distinctive interactions of 5-HT and model membranes could be related to nonspecific effects of this neurotransmitter.