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PDBsum entry 2jcs
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References listed in PDB file
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Key reference
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Title
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Functional studies of active-Site mutants from drosophila melanogaster deoxyribonucleoside kinase. Investigations of the putative catalytic glutamate-Arginine pair and of residues responsible for substrate specificity.
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Authors
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L.Egeblad-Welin,
Y.Sonntag,
H.Eklund,
B.Munch-Petersen.
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Ref.
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Febs J, 2007,
274,
1542-1551.
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PubMed id
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Abstract
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The catalytic reaction mechanism and binding of substrates was investigated for
the multisubstrate Drosophila melanogaster deoxyribonucleoside kinase. Mutation
of E52 to D, Q and H plus mutations of R105 to K and H were performed to
investigate the proposed catalytic reaction mechanism, in which E52 acts as an
initiating base and R105 is thought to stabilize the transition state of the
reaction. Mutant enzymes (E52D, E52H and R105H) showed a markedly decreased
k(cat), while the catalytic activity of E52Q and R105K was abolished. The E52D
mutant was crystallized with its feedback inhibitor dTTP. The backbone
conformation remained unchanged, and coordination between D52 and the dTTP-Mg
complex was observed. The observed decrease in k(cat) for E52D was most likely
due to an increased distance between the catalytic carboxyl group and 5'-OH of
deoxythymidine (dThd) or deoxycytidine (dCyd). Mutation of Q81 to N and Y70 to W
was carried out to investigate substrate binding. The mutations primarily
affected the K(m) values, whereas the k(cat) values were of the same magnitude
as for the wild-type. The Y70W mutation made the enzyme lose activity towards
purines and negative cooperativity towards dThd and dCyd was observed. The Q81N
mutation showed a 200- and 100-fold increase in K(m), whereas k(cat) was
decreased five- and twofold for dThd and dCyd, respectively, supporting a role
in substrate binding. These observations give insight into the mechanisms of
substrate binding and catalysis, which is important for developing novel suicide
genes and drugs for use in gene therapy.
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