TY - JOUR

T1 - Metal-ion dependent catalytic properties of Sulfolobus solfataricus class II α-mannosidase

AU - Nielsen, Jonas Willum

AU - Poulsen, Nina Rødtness

AU - Johnsson, Anna Margit Susanne

AU - Winther, Jakob R.

AU - Stipp, Susan Louise Svane

AU - Willemoës, Martin

PY - 2012

Y1 - 2012

N2 - The active site for the family GH38 class II α-mannosidase is constituted in part by a divalent metal ion, mostly Zn(2+), as revealed in the crystal structures of enzymes from both animal and bacterial sources. The metal ion coordinates to the bound substrate and side chains of conserved amino acid residues. Recently, evidence has accumulated that class II α-mannosidase is active in complex with a range of divalent metal ions. In the present work, with employment of the class II α-mannosidase, ManA, from the hyperthermophilic archaeon Sulfolobus solfataricus, we explored the influence of the divalent metal ion on the associated steady-state kinetic parameters, K(M) and k(cat), for various substrates. With p-nitrophenyl-α-d-mannoside as a substrate, the enzyme showed activity in the presence of Co(2+), Cd(2+), Mn(2+), and Zn(2+), whereas Ni(2+) and Cu(2+) were inhibitory and nonactivating. Co(2+) was the preferred metal ion, with a k(cat)/K(M) value of about 120 mM(-1) s(-1), 6 times higher than that with Cd(2+) and Zn(2+) and 10 times higher than that with Mn(2+). With α-1,2-, α-1,3-, α-1,4-, or α-1,6-mannobiose as a substrate, Co(2+) was the only metal ion promoting hydrolysis of all substrates; however, Mn(2+), Cd(2+), and Zn(2+) could substitute to a varying extent. A change in the divalent metal ion generally affected the K(M) for the hydrolysis of p-nitrophenyl-α-d-mannoside; however, changes in both k(cat) and K(M) for the hydrolysis of α-mannobioses were observed, along with changing preferences for the glycosidic linkage. Finally, it was found that the metal ion and substrate bind in that order via a steady-state, ordered, sequential mechanism.

AB - The active site for the family GH38 class II α-mannosidase is constituted in part by a divalent metal ion, mostly Zn(2+), as revealed in the crystal structures of enzymes from both animal and bacterial sources. The metal ion coordinates to the bound substrate and side chains of conserved amino acid residues. Recently, evidence has accumulated that class II α-mannosidase is active in complex with a range of divalent metal ions. In the present work, with employment of the class II α-mannosidase, ManA, from the hyperthermophilic archaeon Sulfolobus solfataricus, we explored the influence of the divalent metal ion on the associated steady-state kinetic parameters, K(M) and k(cat), for various substrates. With p-nitrophenyl-α-d-mannoside as a substrate, the enzyme showed activity in the presence of Co(2+), Cd(2+), Mn(2+), and Zn(2+), whereas Ni(2+) and Cu(2+) were inhibitory and nonactivating. Co(2+) was the preferred metal ion, with a k(cat)/K(M) value of about 120 mM(-1) s(-1), 6 times higher than that with Cd(2+) and Zn(2+) and 10 times higher than that with Mn(2+). With α-1,2-, α-1,3-, α-1,4-, or α-1,6-mannobiose as a substrate, Co(2+) was the only metal ion promoting hydrolysis of all substrates; however, Mn(2+), Cd(2+), and Zn(2+) could substitute to a varying extent. A change in the divalent metal ion generally affected the K(M) for the hydrolysis of p-nitrophenyl-α-d-mannoside; however, changes in both k(cat) and K(M) for the hydrolysis of α-mannobioses were observed, along with changing preferences for the glycosidic linkage. Finally, it was found that the metal ion and substrate bind in that order via a steady-state, ordered, sequential mechanism.

KW - Bacterial Proteins

KW - Catalytic Domain

KW - Gene Expression Regulation, Bacterial

KW - Gene Expression Regulation, Enzymologic

KW - Metals

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Protein Conformation

KW - Substrate Specificity

KW - Sulfolobus solfataricus

KW - alpha-Mannosidase

U2 - 10.1021/bi301096a

DO - 10.1021/bi301096a

M3 - Journal article

C2 - 22989181

VL - 51

SP - 8039

EP - 8046

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 40

ER -