Human alpha-L-iduronidase. 2. Catalytic properties.
The kinetic parameters of human liver alpha-L-iduronidase were determined with three disaccharide substrates: alpha-L-iduronosyl(1----4)2,5-anhydro-D-[1-3H]mannitol 6-sulphate, alpha-L-iduronosyl(1----4)2,5-anhydro-D-[1-3H]mannitol and alpha-L-iduronosyl(1----3)2,5-anhydro-D-[1-3H]talitol 4-sulphate, derived from the natural substrates heparin and dermatan sulphate and one synthetic, fluorogenic substrate, 4-methylumbelliferyl alpha-L-iduronide. The enzyme activity with all four substrates was optimal at about pH 4.5. The Km values derived using the disaccharide substrates were elevated up to 10-fold with up to a 6.5-fold increase in ionic strength whereas that for the synthetic substrate was only increased by 1.7-fold. The V values for all substrates were unaffected. The inhibitory effect of NaCl, Na2SO4, NaH2PO4 or CuCl2 on enzyme activity was more pronounced with the disaccharide substrates than with the synthetic substrate. The moiety which is most important in binding is the idopyranosyl residue. While the aglycone residue adds to the net affinity for the enzyme, it is the substituent groups of both residues which appear to control catalysis. Specifically the carboxyl moiety of the alpha-L-iduronic acid residue is essential for catalysis while the presence of sulphate on the C4 or C6 position of the aglycone residue has a major influence on catalysis rather than binding. alpha-L-Idosyl(1----4)2,5-anhydro-D-[1-3H]mannitol 6-sulphate did not undergo catalysis and was a potent inhibitor of enzyme activity, whereas beta-glucuronosyl(1----4)2,5-anhydro-D-[1-3H]mannitol 6-sulphate, alpha-L-iduronosyl-2-sulphate(1----4)2,5-anhydro-D-[1-3H]-mannitol 6-sulphate and 4-methylumbelliferyl alpha-L-idoside did not undergo catalysis and were not inhibitory. A model of the catalytic requirements of alpha-L-iduronidase is proposed.
D002384:Catalysis; D003300:Copper / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007068:Iduronidase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008968:Molecular Conformation; D009994:Osmolar Concentration; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013431:Sulfates / Q000494:pharmacology
D013431:Sulfates; C012036:sodium sulfate; D012965:Sodium Chloride; D003300:Copper; D006026:Glycoside Hydrolases; D007068:Iduronidase; C029892:cupric chloride