Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase

Eva Johansson; Jan Neuhard; Martin Willemoës; Sine Larsen

doi:10.1021/bi035893x

Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase

Eva Johansson, Jan Neuhard, Martin Willemoës, Sine Larsen

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

26 Citationer (Scopus)

Abstract

Udgivelsesdato: 2004-May-25

Originalsprog	Engelsk
Tidsskrift	Biochemistry
Vol/bind	43
Udgave nummer	20
Sider (fra-til)	6020-9
Antal sider	9
ISSN	0006-2960
DOI	https://doi.org/10.1021/bi035893x
Status	Udgivet - 2004

Bibliografisk note

Keywords: Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Cytidine Deaminase; Enzyme Stability; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Mutagenesis, Site-Directed; Protein Structure, Quaternary; Protein Subunits; Zinc

Adgang til dokumentet

10.1021/bi035893x

Citationsformater

@article{0c6135706f8e11de8bc9000ea68e967b,

title = "Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase",

abstract = "The zinc-containing cytidine deaminase (CDA, EC 3.5.4.5) is a pyrimidine salvage enzyme catalyzing the hydrolytic deamination of cytidine and 2'-deoxycytidine forming uridine and 2'-deoxyuridine, respectively. Homodimeric CDA (D-CDA) and homotetrameric CDA (T-CDA) both contain one zinc ion per subunit coordinated to the catalytic water molecule. The zinc ligands in D-CDA are one histidine and two cysteine residues, whereas in T-CDA zinc is coordinated to three cysteines. Two of the zinc coordinating cysteines in T-CDA form hydrogen bonds to the conserved residue Arg56, and this residue together with the dipole moments from two alpha-helices partially neutralizes the additional negative charge in the active site, leading to a catalytic activity similar to D-CDA. Arg56 has been substituted by a glutamine (R56Q), the corresponding residue in D-CDA, an alanine (R56A), and an aspartate (R56D). Moreover, one of the zinc-liganding cysteines has been substituted by histidine to mimic D-CDA, alone (C53H) and in combination with R56Q (C53H/R56Q). R56A, R56Q, and C53H/R56Q contain the same amount of zinc as the wild-type enzyme. The zinc-binding capacity of R56D is reduced. Only R56A, R56Q, and C53H/R56Q yielded measurable CDA activity, R56A and R56Q with similar K(m) but decreased V(max) values compared to wild-type enzyme. Because of dissociation into its inactive subunits, it was impossible to determine the kinetic parameters for C53H/R56Q. R56A and C53H/R56Q display increased apparent pK(a) values compared to the wild-type enzyme and R56Q. On the basis of the structures of R56A, R56Q, and C53H/R56Q an explanation is provided of kinetic results and the apparent instability of C53H/R56Q.",

author = "Eva Johansson and Jan Neuhard and Martin Willemo{\"e}s and Sine Larsen",

note = "Keywords: Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Cytidine Deaminase; Enzyme Stability; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Mutagenesis, Site-Directed; Protein Structure, Quaternary; Protein Subunits; Zinc",

year = "2004",

doi = "10.1021/bi035893x",

language = "English",

volume = "43",

pages = "6020--9",

journal = "Biochemistry",

issn = "0006-2960",

publisher = "American Chemical Society",

number = "20",

}

TY - JOUR

T1 - Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase

AU - Johansson, Eva

AU - Neuhard, Jan

AU - Willemoës, Martin

AU - Larsen, Sine

N1 - Keywords: Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Cytidine Deaminase; Enzyme Stability; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Mutagenesis, Site-Directed; Protein Structure, Quaternary; Protein Subunits; Zinc

PY - 2004

Y1 - 2004

N2 - The zinc-containing cytidine deaminase (CDA, EC 3.5.4.5) is a pyrimidine salvage enzyme catalyzing the hydrolytic deamination of cytidine and 2'-deoxycytidine forming uridine and 2'-deoxyuridine, respectively. Homodimeric CDA (D-CDA) and homotetrameric CDA (T-CDA) both contain one zinc ion per subunit coordinated to the catalytic water molecule. The zinc ligands in D-CDA are one histidine and two cysteine residues, whereas in T-CDA zinc is coordinated to three cysteines. Two of the zinc coordinating cysteines in T-CDA form hydrogen bonds to the conserved residue Arg56, and this residue together with the dipole moments from two alpha-helices partially neutralizes the additional negative charge in the active site, leading to a catalytic activity similar to D-CDA. Arg56 has been substituted by a glutamine (R56Q), the corresponding residue in D-CDA, an alanine (R56A), and an aspartate (R56D). Moreover, one of the zinc-liganding cysteines has been substituted by histidine to mimic D-CDA, alone (C53H) and in combination with R56Q (C53H/R56Q). R56A, R56Q, and C53H/R56Q contain the same amount of zinc as the wild-type enzyme. The zinc-binding capacity of R56D is reduced. Only R56A, R56Q, and C53H/R56Q yielded measurable CDA activity, R56A and R56Q with similar K(m) but decreased V(max) values compared to wild-type enzyme. Because of dissociation into its inactive subunits, it was impossible to determine the kinetic parameters for C53H/R56Q. R56A and C53H/R56Q display increased apparent pK(a) values compared to the wild-type enzyme and R56Q. On the basis of the structures of R56A, R56Q, and C53H/R56Q an explanation is provided of kinetic results and the apparent instability of C53H/R56Q.

AB - The zinc-containing cytidine deaminase (CDA, EC 3.5.4.5) is a pyrimidine salvage enzyme catalyzing the hydrolytic deamination of cytidine and 2'-deoxycytidine forming uridine and 2'-deoxyuridine, respectively. Homodimeric CDA (D-CDA) and homotetrameric CDA (T-CDA) both contain one zinc ion per subunit coordinated to the catalytic water molecule. The zinc ligands in D-CDA are one histidine and two cysteine residues, whereas in T-CDA zinc is coordinated to three cysteines. Two of the zinc coordinating cysteines in T-CDA form hydrogen bonds to the conserved residue Arg56, and this residue together with the dipole moments from two alpha-helices partially neutralizes the additional negative charge in the active site, leading to a catalytic activity similar to D-CDA. Arg56 has been substituted by a glutamine (R56Q), the corresponding residue in D-CDA, an alanine (R56A), and an aspartate (R56D). Moreover, one of the zinc-liganding cysteines has been substituted by histidine to mimic D-CDA, alone (C53H) and in combination with R56Q (C53H/R56Q). R56A, R56Q, and C53H/R56Q contain the same amount of zinc as the wild-type enzyme. The zinc-binding capacity of R56D is reduced. Only R56A, R56Q, and C53H/R56Q yielded measurable CDA activity, R56A and R56Q with similar K(m) but decreased V(max) values compared to wild-type enzyme. Because of dissociation into its inactive subunits, it was impossible to determine the kinetic parameters for C53H/R56Q. R56A and C53H/R56Q display increased apparent pK(a) values compared to the wild-type enzyme and R56Q. On the basis of the structures of R56A, R56Q, and C53H/R56Q an explanation is provided of kinetic results and the apparent instability of C53H/R56Q.

U2 - 10.1021/bi035893x

DO - 10.1021/bi035893x

M3 - Journal article

C2 - 15147186

VL - 43

SP - 6020

EP - 6029

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 20

ER -