Mechanistic Investigation of a Starch-Branching Enzyme Using Hydrodynamic Volume SEC Analysis

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Abstract
Two linear α-(1,4)-d-glucans substrates, of degrees of polymerization DP ∼ 150 and 6000, were exposed to maize starch-branching enzyme IIa (mSBEIIa) in vitro. The resulting branched α-glucans and their constituent chains (obtained by debranching) were analyzed by nuclear magnetic resonance (NMR) and size-exclusion chromatography (SEC). SEC data for the debranched species are presented as chain-length distributions, while those for branched species are presented as hydrodynamic volume distributions (HVDs), which is the most meaningful way to present such data (because SEC separates by size, not molar mass, and a sample of branched polymers with the same size can have a range of molar masses). A rigorous interpretation of the HVDs of the substrate and its branched product show that at least part of the branching is an interchain transfer mechanism in both the short- and long-chain substrate cases. A bimodal HVD of the in vitro branched α-glucan derived from the short-chain substrate was observed, and it is postulated that the divergence of the two populations is due to very small chains being unable to undergo branching. In the case of the in vitro branching of the long-chain substrate, the formation of maltohexaose during the reaction and the presence of a monomodal HVD were observed, suggesting a distinct mode of action of mSBEIIa on this substrate. Quantification of the branching level by NMR showed the branched glucans from both substrates had substantial amounts of branching (2.1–4.5%), ascribed to the intrinsic nature of the action of mSBEIIa on the two substrates. It is postulated that differences in the degrees of substrate association affect the pattern of branching catalyzed by the enzyme, and a putative active site structure is proposed based on the appearance of maltohexaose. The molar mass distribution of the constituent chains of the in vitro branched α-glucans obtained by isoamylase treatment reveals the transfer of chains of specific size and supports the supposition given in the literature that mSBEIIa is responsible for short-chain branching in amylopectin. It is suggested that hydrodynamic volume SEC analysis should be used as a tool for the mechanistic investigation of SBEs, allowing SEC data of in vitro branched α-glucans to be both comparable and quantitative.