科研之友
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摘要
This study was undertaken to examine the mechanistic significance of two highly conserved residues positioned in the active site of pyruvate dehydrogenase kinase, Glu-243 and His-239. We used site-directed mutagenesis to convert Glu-243 to Ale, Asp, or Gin and His-239 to Ala. The resulting mutant kinases demonstrated a greatly reduced capacity for phosphorylation of pyruvate dehydrogenase, The Glu-243 to Asp mutant had similar to2% residual activity, whereas the Glu-243 to Ala or Gin mutants exhibited less than 0.5 and 0.1% residual activity, respectively. Activity of the His-239 to Ala mutant was decreased by similar to 90%. Active-site titration with [alpha-P-32]ATP revealed that neither Glu-243 nor His-239 mutations affected nucleotide binding. All mutant kinases showed similar or even somewhat greater affinity than the wild-type kinase toward the protein substrate, pyruvate dehydrogenase complex. Furthermore, neither of the mutations affected the inter-subunit interactions. Finally, pyruvate dehydrogenase kinase was found to possess a weak ATP hydrolytic activity, which required Glu-243 and His-239 similar to the kinase activity. Based on these observations, we propose a mechanism according to which the invariant glutamate residue (Glu-243) acts as a general base catalyst, which activates the hydroxyl group on a serine residue of the protein substrate for direct attack on the gamma phosphate. The glutamate residue in turn might be further polarized through interaction with the neighboring histidine residue (His-239).
- 出版日期2001-5-25
