Journal article
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 KM for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, KA, 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
Language: | English |
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Year: | 2015 |
Pages: | 2032-2039 |
ISSN: | 15204995 and 00062960 |
Types: | Journal article |
DOI: | 10.1021/acs.biochem.5b00090 |
ORCIDs: | Harris, Pernille , 0000-0001-6995-9154 and 0000-0003-1689-2712 |
4-nitrophenyl-alpha-D-mannopyranoside Amino Acid Substitution Archaeal Proteins Cadmium Catalytic Domain Cations, Divalent Cobalt Enzyme Stability Hydrogen-Ion Concentration Isoenzymes Kinetics Ligands Manganese Mannosides Metals Mutant Proteins Osmolar Concentration Recombinant Proteins Sulfolobus solfataricus Zinc alpha-Mannosidase