Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from Sulfolobus solfataricus
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Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from Sulfolobus solfataricus. / Hansen, Dennis; Webb, Helen; Nielsen, Jonas Willum; Harris, Pernille; Winther, Jakob R.; Willemoës, Martin.
I: Biochemistry, Bind 54, Nr. 11, 2015, s. 2032-2039.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from Sulfolobus solfataricus
AU - Hansen, Dennis
AU - Webb, Helen
AU - Nielsen, Jonas Willum
AU - Harris, Pernille
AU - Winther, Jakob R.
AU - Willemoës, Martin
PY - 2015
Y1 - 2015
N2 - 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 (Co(2+), Zn(2+), Mn(2+), or Cd(2+)). 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 Zn(2+) 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.
AB - 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 (Co(2+), Zn(2+), Mn(2+), or Cd(2+)). 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 Zn(2+) 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.
U2 - 10.1021/acs.biochem.5b00090
DO - 10.1021/acs.biochem.5b00090
M3 - Journal article
C2 - 25751413
VL - 54
SP - 2032
EP - 2039
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 11
ER -
ID: 138274840