Human alpha-L-iduronidase. 2. Catalytic properties

Abstract

1. The kinetic parameters of human liver a-L-iduronidase were determined with three disaccharide substrates: a-L-iduronosyl(l+4)2,5-anhydro-~-[l-~H]mannitol 6-sulphate, a-i,-iduronosyl(1+4)2,5-anhydro-~-[l-~H]mannitol and a-L-iduronosyl(1 + 3)2,5-anhydro-u-[l -3H]talitol 4-sulphate, derived from the natural substrates heparin and dermatan sulphate and one synthetic, fluorogenic substrate, 4-methylumbelliferyl a-L-iduronide.2. The enzyme activity with all four substrates was optimal at about pH 4.5. The K, values derived using the disaccharide substrates were elevated up to 10-fold with u p 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, Na2S04, NaH2P04 or CuCI, on enzyme activity was more pronounced with the disaccharide substrates than with the synthetic substrate.3. The moiety which is most important in binding is the idopyranosyl residue. While the aglycone residue adds to the nett affinity for the enzyme, it is the substituent groups of both residues which appear to control catalysis. Specifically the carboxyl moiety of the a-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. a-L-Idosyl(l-+4)2,5-anhydro-~-[l-~H]mannitol6-sulphate did not undergo catalysis and was a potent inhibitor of enzyme activity, whereas /?-glucuronosyl(1+4)2,5-anhydro-u-[1 -3H]mannitol 6-sulphate, ELiduronosyl-2-sulphate(l+4)2,5-anhydro-~-[l-~H]-mannitol 6-sulphate and 4-methylumbelliferyl a-L-idoside did not undergo catalysis and were not inhibitory. . A model of the catalytic requirements of a-L-iduronidase is proposed.sr-L-Iduronidase (a-L-iduronide iduronohydrolase) is one of many lysosomal hydrolases involved in the degradation of the sulphated glycosaminoglycans heparan sulphate (HS) and dermatan sulphate (DS) [l]. Deficiency of this enzyme activity in humans leads to the accumulation of HS and DS fragments with non-reducing-end a-I,-iduronosyl residues and leads to the disease state described as mucopolysaccharidosis 1 (MPS the I ) [l]. The disease can range in clinical symptoms from severe (MPS, IH, Hurler Syndrome) to mild (MPS IS, Scheie syndrome) and all shades in between. This phenotypic variation cannot yet be predicted from biochemical data and it has not yet been established whether the variation results from an absencc of enzyme protein or inactive or partially active 6-sulphate; Ido-anM6S, a-I.-idosyl(l-t4)2,5-anhydro-u-[I -3 H]mannitol6-sulphate; IdoA-4MeUmb, 4-methylumbelliferyl a-1.-iduronide; Ido-4MeUmb, 4-methylumbelliferyl a-i-idoside; GlcNAc, N-acctyl-mglucosamine; GlcNAc6S, N-acctyl-u-glucosamine 6-sulphate; NaMe2Glt, sodium dimethylglutarate; HS, heparan sulphate; DS, dermatan sulphate. ~-Enzjrne. a-1,-Iduronidase (EC 3.2.1.76).protein. Understanding the catalytic properties of a-Liduronidase toward its natural substrates is an impor...

Human liver sulphamate sulphohydrolase. Determinations of native protein and subunit Mr values and influence of substrate agylcone structure on catalytic properties

“…In preliminary studies of the effect of ionic strength and buffer pH on subunit aggregation we were unable to alter the enzyme's native protein Mr. This result is different from a finding we have reported for z-L-iduronidase, another lysosomal enzyme involved in heparan sulphate degradation, in which buffer pH and ionic strength influenced subunit aggregation (Clements et al, 1985a). We reported previously that human liver sulphamate sulphohydrolase has a native protein Mr of 190000 and that higher enzymically inactive aggregates were also present (Mahuran et al, 1983a).…”

“…Finally,-we have suggested that some or all of the eight enzymes involved in the degradation of the complex structured substrateheparan sulphatecouldby association with each other form an enzyme cluster (Clements et al, 1985a). This type of association would be expected to improve considerably the overall catalytic efficiency of the system by facilitated diffusion of the product of one enzyme that becomes the substrate for the next enzyme (Wombacher, 1983).…”

Clinical implementation of gene panel testing for lysosomal storage diseases

“…The biochemical confirmation was performed both in external accredited laboratories or in-house. In-house confirmation was done for IDUA (Anson, Bielicki, & Hopwood, 1992;Clements, Muller, & Hopwood, 1985), GAA (Beratis, LaBadie, & Hirschhorn, 1978) and GBA (Beutler & Kuhl, 1970). Briefly, for IDUA and GBA activity measurements, peripheral blood leukocytes were used and were extracted by adding 2 ml of a 2% dextran solution to 5 ml of blood sample.…”

Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase

“…The fluorogenic substrate 4-methylumbelliferyl 2-acetamido-2deoxy-,/-D-glucopyranoside was used to measure fl-hexosaminidase activity [14]. Iduronate-2-sulphatase (IDS) [15], a-L-Iduronidase [16] and N-acetylgalactosamine-4-sulphatase (4S) [17] were assayed by previously reported procedures.…”