3bsqA Discussion

Multiple sequence alighment (MSA) revealed several conserved residues throughput the whole sulfatase family; more of them in the N-terminal side of the sequence. As mentioned in introduction, ASA and ASB are lysosomal enzymes while ASC is a microsomal enzyme. Litrerature search revealed that arylsulfatases D, E, F, G, H, J and K are localized in ER and golgi compartments of the cell N-acetylgalascosamine -4- sulfatase, ASA and STS were shown to be most similar in structure to ASK, by DALI results. Therefore the literature was searched to fine the mechanism of catalytic action of these enzymes. Only STS resulted a complete experimental determination of the function. This study describes a set of nine residues which are essential for STS function. ([[1]] [1]). However STS is a membrane bound protein which consists of a globular domain bearing the catalytic site and a transmembrane domain made up of two antiparallel hydrophobic alpha helices. The three dimentional stucture of ASK is not indicative of a transmembrane domain; therefore it may be a water soluble enzyme found in ER [1].

Evidence from previous experiments states that ASA is localysed in lysosomes [2], which is very different  in pH environment to ER and golgi. Therefore STS was chossen to study the structure and function of the catalytic site. There are nine key catalytic residues in STS; which are D35, D36, D342, G343, C75, R79, K134, K368, H136 and H290. First four residues provide oxygen ligands for the divalent cation bindin while thers participate in hydrolysis of the substrate. It should be noticed that C is post translationally modified to a formyleglycine (FG), which is further oxydised to a hydroxyformyleglycine (HFG), to act as a potent nucleophile towards the carbon center which bonds with the sulphur in the substrate.

MSA showed that these STS catalytic residues are conserved generally all through the enzyme family and especially in ASA and STS. These residues were marked on the three-dimentioal structure of ASK on PyMol, they made up a very similar catalytic site to that of STS. Five out of nine STS key residues were located on exactly the same position of the sequence; however D36 and C75 seemd to have substituted with H and S, while K368 occurred a few positions earlier and H290 was lost. When these residues were marked in the 3D-structure, a H was found in close proximity to the catalytic site and in very similar position to that of H290, but occurred at H284 position of ASK sequence. It is marked in ‘cyan’ on figure 2. The STS-K368-like residue in ASK (K296) seemed to have conserved all throught the MSA. As mentionaed earlier, there are four residues which provide electronegative oxygen ligands to hold the bivalent cation in the catalytic site. Three of these residues (D24, D283 and G284) are conserved in ASK, but D36 in STS is replaced by a H 25 in ASK. However considering that one of the Nitrogens on H side-chain ring could donate a lonepair to form a coordinate bond with ‘the divalent cation’, this region may still function similarly. Finally, the C75 of STS being substituted by a serine (S) in the bacterial ASK (this is conserven in the human ASK sequence) could also be explained by a possible post translational modification of S to form a FG. The crystal structure of ASK doesn’t show this modification; however due to highly similar structures of S and FG, under 2.4 Å resolution, may not have distinguished the difference between HO-C (1.4 Å ) and O=C (1.2 Å) bonds. A study done on ‘’Klebsiella pneumoneae’’ shows that part of the ASK polypeptides in the cell have FG in the place of S by a different post translational modification mechanism to that of eukaryotes [[2]].

C-terminus of sulfatases are recognised to contain a substrate binding site, hence weakly conserved throughout the family due to the variation of types of substrates used. However, when the C-terminal regions in two sequence alignment of ASK and STN was looked at, the level of conservation was very high. Based on this evidence it is predicted, that STS and ASK shair substrates.

Possible function and likely substrates of ASK 

STS is a membrane bound enzyme, mostly found in human placenta and skin fibroblasts. Itconverts sex-stroid precursors to ative estrogen and androgen, thereofre give a local suppley of these hormones (Ghosh, D., 2007). ASK is a water soluble enzyme localysed in ER of the cell. No tissue localization is specified to the data. however, Based on all the above evidence, the possible function ASK may be binding and activation of post translationally modified stroids in the ER lumen.