Quaternary structure of human nucleoside diphosphate kinase isoforms HA and HB in solution

Schaertl, Sabine

doi:10.1016/0014-5793(96)00978-7

Cited by 11 publications

(6 citation statements)

References 27 publications

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“…Other mammalian NDP kinases behave abnormally in size‐exclusion chromatography. For example, NDP kinase B was claimed to be a tetramer [34] but other studies in solution [9] and crystallographic data indicate a hexameric structure [35,36]. We assume that the aberrant elution from the size exclusion column is due to interaction of the protein with the gel matrix, and not to dissociation of the subunits.…”

Section: Resultsmentioning

confidence: 99%

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene

Erent

Gonin

Cherfils

et al. 2001

European Journal of Biochemistry

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The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CD. NDP kinase CD had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CD, based on the crystal structure of NDP kinase B, indicated that NDP kinase CD had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CD readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.

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Section: Resultsmentioning

confidence: 99%

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene

Erent

Gonin

Cherfils

et al. 2001

European Journal of Biochemistry

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“…The cell debris was pelleted by centrifugation for 1 h at 11,000 rpm in an SS-34 rotor in a Sorvall RC-5B. Unmodified proteins NDK-A and -B were purified and characterized as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

Substrate Specificity of Human Nucleoside-diphosphate Kinase Revealed by Transient Kinetic Analysis

Schaertl

Konrad

Geeves³

1998

Journal of Biological Chemistry

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Nucleoside-diphosphate kinases (NDKs) catalyze the transfer of ␥-phosphoryl groups from NTPs via an active site histidine to NDPs using a ping-pong mechanism. We have used the change of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and dephosphorylation with a range of nucleotides and nucleotide analogues. For natural nucleotides, the rates of phosphorylation and dephosphorylation were linearly dependent upon nucleotide concentration until they became too fast to measure. The second order rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 ؋ 10 6 M ؊1 s ؊1 . Dephosphorylation by NDPs was 2-3-fold faster than the corresponding phosphorylation reaction, and dephosphorylation by dNDPs was 3-4-fold slower than the equivalent NDPs. In all cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes the transfer of thiophosphate from adenosine 5-O-(thiotriphosphate) (ATP␥S) and guanosine 5-O-(thiotriphosphate) (GTP␥S) to NDP, but at 1 ⁄1000 of the equivalent phosphoryl transfer rates. In this case, the observed rate constants of phosphorylation and dephosphorylation were hyperbolically dependent on nucleotide concentration. Thiophosphorylation by ATP␥S and GTP␥S occurred with k max of 2.8 and 1.35 s ؊1 and K d of 145 and 36 M respectively. For dethiophosphorylation by a range of NDPs, k max was in the range of 5-30 s ؊1 , whereas K d varied between 0.16 and 3.3 mM. Guanine had the lowest K d values, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting phosphoryl transfer. Thiophosphates change only the rate of the phosphoryl transfer step, whereas both events are influenced by the base. Modification at the 2-hydroxyl of ribose has only a small effect, while the overall rate of phosphoryl transfer is reduced 1000-fold by modification at the 3-ribose.Nucleoside-diphosphate kinase (NDK 1 ; EC 2.7.4.6) is a ubiquitous enzyme that catalyzes the transfer of the ␥-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate (1), involving a phosphohistidine intermediate state (2). NDK exists as either hexamers (most eukaryotic species) or tetramers (most prokaryotic species) and has been reported to display little specificity for different nucleotide bases (2, 3), reflecting a pivotal role in maintaining balanced levels of all oxy-and deoxynucleoside triphosphates in the cell. It is thus considered a housekeeping enzyme (4), a role that has taken on greater significance with the therapeutic use of nucleotide analogues such as azidothymidine (AZT) as replication inhibitors as these compounds must be phosphorylated in the cell before becoming active (5).In humans, two very closely (89% identity) related isoforms of NDK, designated NDK-A and NDK-B, were identified biochemically (6), which associate in vivo to form mixed hexameric isoenzymes. Evidence that NDK-A and -B may...

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“…The phosphorylated proteins was stable for several hours when kept on ice. Protein concentrations were estimated by using an extinction coefficient of 1.3 cm 2 mg Ϫ1 at 280 nm (18) and molecular masses of 17.1 kDa for NDK-A and 17.2 kDa for NDK-B and are given with respect to catalytic sites, i.e. monomers.…”

Section: Methodsmentioning

confidence: 99%

“…Proteins were analyzed by 15% SDS-polyacrylamide gel electrophoresis (19). Gel filtration was performed in buffer A on a Superose 6 column on an Amersham Pharmacia Biotech FPLC apparatus as described before (18).…”

Section: Methodsmentioning

confidence: 99%

Human Nucleoside Diphosphate Kinase B (Nm23-H2) from Melanoma Cells Shows Altered Phosphoryl Transfer Activity Due to the S122P Mutation

Schaertl

Geeves

Konrad

1999

Journal of Biological Chemistry

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) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40 -200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the ratelimiting step of the NDK-B S122P -catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser 120 3 Gly mutation in human NDK-A/ Nm23-H1, the Ser 122 3 Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.

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Quaternary structure of human nucleoside diphosphate kinase isoforms HA and HB in solution

Cited by 11 publications

References 27 publications

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene

Substrate Specificity of Human Nucleoside-diphosphate Kinase Revealed by Transient Kinetic Analysis

Human Nucleoside Diphosphate Kinase B (Nm23-H2) from Melanoma Cells Shows Altered Phosphoryl Transfer Activity Due to the S122P Mutation

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