Interaction of arylsulfatase-A (ASA) with its natural sulfoglycolipid substrates: a computational and site-directed mutagenesis study

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作者：

M Schenk，CAK Koppisetty，DC Santos，E Carmona，S Bhatia，PG Nyholm，N Tanphaichitr

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摘要：

Arylsulfatase A (ASA) hydrolyzes sulfate esters with a pH optimum of 5. Interactions between p-nitrocatechol sulfate (NCS, artificial substrate) and active site residues of ASA are revealed from their co-crystal structure. Since equivalent ASA interactions with its natural substrates, sulfogalactosylceramide (SGC) and sulfogalactosylglycerolipid (SGG), are not known, we computationally docked SGC/SGG to the ASA crystal structure. Our dockings suggested that Cys69 was the active site residue, and Lys302 & Lys123 as residues anchoring the sulfate group of SGC/SGG to the active site, as observed for NCS. We further confirmed these results using 2 recombinant ASA mutants: C69A and CKK (Cys69, Lys302 and Lys123-all mutated to Ala). Both ASA mutants failed to desulfate SGC/SGG, and CKK showed minimal binding to [(14)C]SGC, although C69A still had affinity for this sulfoglycolipid. However, our dockings suggested additional intermolecular hydrogen bonding and hydrophobic interactions between ASA and SGC/SGG, thus contributing to the specificity of SGC/SGG as natural substrates.

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关键词：

Sulfatase Arylsulfatase-A Sulfogalactosylglycerolipid Sulfogalactosylceramide Galactosyl sulfate Structural docking pK a calculation Enzyme–substrate interaction