Enzymatic Characteristics, Biological Substrate And Catalytic Mechanism Of Human Hex D

Glycosylation is an important post-translational modification, and participate in manycell activities, such as signal transduction and cell recognition. Glycosidase is an enzymehydrolysis of the glycosidic bond，the hydrolysis of glycoconjugates is an essential part of themetabolism of organisms. Glycosylation metabolic abnormalities is closely related with manydiseases, such as Tay-Sachs disease, Sandhoff disease, Ⅱdiabetes, alzheimer disease, andcancer. Human hexosaminidase D （HexD） is a new glucoside hydrolase, and its substratespecificity and catalytic mechanism are both unknown.The cDNA of HexD was cloned to the vector pET3C, and then the recombinant plasmidwas transformed to E.Coli BL21（DE3） plysS. The expressed protein was purified by Ni-NTAcolumn. Then the optimum pH, thermo stability, and the influence of metal ions weredetermined. At the same time，use of biological information to analyze the protein sequencesof the same family of enzymes find that Asp148and the Glu149was the two key amino acids. Then, the HexD hydrolysis of a variety of substrates, such as fluorescent substrates,peptides,and glycolipid. The HexD and the other two the hexosaminidase enzyme HexA/HexB andOGA contrast, analysis of the similarities and differences between the three substratespecificity. Finally, expression the purified HexD protein used for the preparation ofpolyclonal antibody. Specific results are as follows.We successfully constructed the pET3C/HexD, and got enough soluble enzyme. Theenzyme properties were determined with4-MU-O-GalNAc as the substrate. The optimum pHand temperature are5.5and37℃respectively. The enzyme is more thermostable, and stillhas high activity after treated0.5hr in50℃. The activity has no significant changes whentreated with1mM metal ions（CuSO₄、FeSO₄·7H₂O、MgCl₂·6H₂O、CaCl₂、NiSO₄·6H₂O、AlCl₃·6H₂O、ZnSO₄·7H₂O、MnCl₂）, but it can be inhibited by10mM AlCl₃, CuSO₄, andFeSO₄·7H₂O, especially Al³⁺. Site-directed mutagenesis were performed at Asp148andGlu149, the enzyme will lost activity when asp148was changed to Glu or Asn, but the changes of Glu149to Asp or Gln have no significant influence to the enzyme. So Asp148andGlu149are the key catalytic residues of HexD. By their biological substrate experiments, wefound HexD prefer O-linked N-Acetyl-β-D-galactose, but not hydrolyze glycolipids. Inaddition, the use of E. coli expression the purified HexD protein. Western blotting analysisshowed that the antibody is the HexD specificity. The HexA/HexB in the lysosomes canhydrolysis O-β-GalNAc structure is also capable of hydrolyzing a substrate for O-β-GlcNAcstructure; OGA in the nucleus and cytoplasm only capable of hydrolyzing O-β-GlcNAcsubstrate.