Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from Sulfolobus solfataricus.
Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the -1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in K(M) for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, K(A), for the divalent metal ion (Co²⁺, Zn²⁺, Mn²⁺, or Cd²⁺). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn²⁺ concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme, although less dramatically with some activating metal ions. No major differences in the pH dependence between wild-type and mutant enzymes were found in the presence of different metal ions. The pH optimum was 5, but enzyme instability was observed at pH <4.5; therefore, only the basic limb of the bell-shaped pH profile was analyzed.
†Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark.
Dennis K
DK
Hansen
†Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark.
Helen
H
Webb
†Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark.
Jonas Willum
JW
Nielsen
‡Department of Chemistry, Technical University of Denmark, Building 206, DK2800 Kgs. Lyngby, Denmark.
Pernille
P
Harris
†Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark.
Jakob R
JR
Winther
†Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark.
Martin
M
Willemoës
D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008362:Mannosides / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism; D043323:alpha-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D002413:Cations, Divalent; D007527:Isoenzymes; D008024:Ligands; D008362:Mannosides; D008670:Metals; D050505:Mutant Proteins; D011994:Recombinant Proteins; D002104:Cadmium; C002731:4-nitrophenyl-alpha-D-mannopyranoside; D003035:Cobalt; D008345:Manganese; D043323:alpha-Mannosidase; D015032:Zinc