Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from Sulfolobus solfataricus

TY - JOUR

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 - 2015/3/24

Y1 - 2015/3/24

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

SN - 0006-2960

VL - 54

SP - 2032

EP - 2039

JO - Biochemistry

JF - Biochemistry

IS - 11

ER -