Diagnostic enzymology of α-L-iduronidase with special reference to a sulphated disaccharide derived from heparin

Iduronosyl anhydro[1-³H]mannitol 6-sulphate (IMs), iduronosyl anhydro[1-³H]mannitol, phenyl iduronide (PhI) and 4-methylumbelliferyl iduronide have been compared as substrates for the diagnostic estimation of α-L-iduronidase activity present in human leucocyte and cultures skin fibroblast homogenates. The pH profile of leucocyte and fibroblast iduronidase activity was dependent on substrate structure and concentration, the ionic strength and the nature of the buffer ion used in the assay mixture. NaCl, KBr and Na₂SO₄ were shown to be parabolic competitive inhibitors of IMs activity, the K(i) with fibroblast homogenates being 34, 13.4 and 0.22 mmol/l respectively. NaCl and KBr were shown to have a primary salt effect on the interaction between enzyme and substrate but Na₂SO₄ appeared to have a specific ion effect at a cationic binding site. NaCl inhibited the hydrolysis of IMs at all pH values studied, whereas NaCl concentrations of 0.2 mol/l inhibited the hydrolysis of PhI and pH values below 3.8 but activated the enzyme at higher incubation pH values. Cu²⁺ was shown to be a potent noncompetitive inhibitor of IMs enzyme activity with an apparent K(i) of approximately 0.02 mmol/l. The enzyme activity was inhibited by Fe²⁺ (K(i) 4 mmol/l), Hg²⁺ and Ag⁺, but has not significantly been affected by other univalent or bivalent cations. The presence of solvent and salt effects on apparent K(m) but not the V(max) suggest that the binding of IMs to the enzyme involved charge neutralization, and it is inferred that two cationic binding sites are present at the active site. It is postulated that one site specifically binds to the iduronic acid carboxyl group, the other to the 6-sulphate of the anhydromannitol moiety.