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. 1999 Nov 15;344(Pt 1):47–53.

Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily.

M Bowker-Kinley 1, K M Popov 1
PMCID: PMC1220612  PMID: 10548532

Abstract

In this study the roles of invariant Asn-247, Asp-282, Gly-284, Gly-286 and Gly-319 of pyruvate dehydrogenase kinase were investigated by site-directed mutagenesis. Recombinant kinases, wild-type, Asn-247Ala, Asp-282Ala, Gly-284Ala, Gly-286Ala and Gly-319Ala, were expressed in bacteria, purified, and characterized. Three mutant kinases, Asn-247Ala, Asp-282Ala and Gly-286Ala, lacked any appreciable activity. Two other mutants, Gly-284Ala and Gly-319Ala, were catalytically active, with apparent V(max) values close to that of the wild-type kinase (67 and 85 versus 70 nmol/min per mg, respectively). The apparent K(m) value of Gly-319Ala for nucleotide substrate increased significantly (1500 versus 16 microM). In contrast, Gly-284Ala had only a slightly higher K(m) value than the wild-type enzyme (28 versus 16 microM). ATP-binding analysis showed that Asn-247Ala, Asp-282Ala and Gly-286Ala could not bind nucleotide. The K(d) value of Gly-284Ala was slightly higher than that of the wild-type enzyme (7 versus 4 microM, respectively). In agreement with kinetic analysis, the Gly-319Ala mutant bound ATP so poorly that it was difficult to determine the binding constant. Despite the fact that Asn-247Ala, Asp-282Ala and Gly-286Ala lacked enzymic activity, they were still capable of binding the protein substrate, as shown by their negative-dominant effect in the competition assay with the wild-type kinase. The results of CD spectropolarimetry indicated that there were no major changes in the secondary structures of Asp-282Ala and Gly-286Ala. These results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus. Furthermore, the catalytic domain is likely to be folded similarly to the catalytic domains of the members of ATPase/kinase superfamily [molecular chaperone heat-shock protein 90 (Hsp90), DNA gyrase B and histidine protein kinases].

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Selected References

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