Phosphorylation sites and inactivation of branched-chain α-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle

Paxton, Ralph; Kuntz, Martha J.; Harris, Robert A.

doi:10.1016/0003-9861(86)90108-6

Cited by 68 publications

(20 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is known that phosphorylation of site 2, serine 303 in rat E1␣, is silent with respect to regulation of BCKDH activity (3)(4)(5). Furthermore, mutations of serine 293, the phosphorylation site 1, result in kinetically altered enzymes (5).…”

Section: Discussionmentioning

confidence: 99%

“…This regulation is primarily exerted through reversible phosphorylation of the E1␣ subunit by BCKDH kinase. BCKDH kinase phosphorylates two serine residues on the E1␣ subunit, although inactivation of the dehydrogenase results exclusively from the phosphorylation of site 1, serine 293 in rat E1␣ (3)(4)(5). Furthermore, phosphorylation of site 2 occurs at a significantly slower rate (3)(4)(5), suggesting that the site 2 serine, serine 303 in rat E1␣, is a poor substrate either due to the primary or secondary structure surrounding this residue or the positioning of this serine residue relative to the kinase active site.…”

mentioning

confidence: 99%

“…BCKDH kinase phosphorylates two serine residues on the E1␣ subunit, although inactivation of the dehydrogenase results exclusively from the phosphorylation of site 1, serine 293 in rat E1␣ (3)(4)(5). Furthermore, phosphorylation of site 2 occurs at a significantly slower rate (3)(4)(5), suggesting that the site 2 serine, serine 303 in rat E1␣, is a poor substrate either due to the primary or secondary structure surrounding this residue or the positioning of this serine residue relative to the kinase active site. BCKDH kinase is regulated in part through inhibition by branched-chain ␣-ketoacids and thiamine pyrophosphate, all of which are substrates for the BCKDH-catalyzed reaction (6,7).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Roles of Amino Acid Residues Surrounding Phosphorylation Site 1 of Branched-chain α-Ketoacid Dehydrogenase (BCKDH) in Catalysis and Phosphorylation Site Recognition by BCKDH Kinase

Hawes

Schnepf

Jenkins

et al. 1995

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Branched-chain ␣-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1␣ subunit. Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched-chain ␣-ketoacid dehydrogenase kinase. Alanine substitution of serine 293 resulted in a 10-fold increased K m for ␣-ketoisovalerate, a less increased (2.8-fold) K m for ␣-ketoisocaproate, but no change in V max or the K m for thiamine pyrophosphate. Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among ␣-ketoacid dehydrogenases, resulted in inactive enzymes. Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1. Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate. Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain ␣-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation. Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiamine pyrophosphate.The activity state of mammalian branched-chain ␣-ketoacid dehydrogenase (BCKDH) 1 is highly regulated according to the physiological requirements for either degradation or conservation of branched-chain amino acids derived from cellular and dietary protein (1, 2). This regulation is primarily exerted through reversible phosphorylation of the E1␣ subunit by BCKDH kinase. BCKDH kinase phosphorylates two serine residues on the E1␣ subunit, although inactivation of the dehydrogenase results exclusively from the phosphorylation of site 1, serine 293 in rat E1␣ (3-5). Furthermore, phosphorylation of site 2 occurs at a significantly slower rate (3-5), suggesting that the site 2 serine, serine 303 in rat E1␣, is a poor substrate either due to the primary or secondary structure surrounding this residue or the positioning of this serine residue relative to the kinase active site. BCKDH kinase is regulated in part through inhibition by branched-chain ␣-ketoacids and thiamine pyrophosphate, all of which are substrates for the BCKDH-catalyzed reaction (6, 7). Inhibition of the kinase by branched-chain ␣-ketoacids is believed to occur in vivo, resulting in a fully active BCKDH in the liver of rats fed a high protein diet (2). However, the exact molecular basis for these inhibitory effects is not clearly understood. BCKDH kinase has also been reported to be activated by its interaction with the E2 subunit of the BCKDH complex, although the molecular basis for this mode of regulation is also not understood (8, 9). Another important feature of BCKDH kinase is its high degree of substrate specificity, resulting in phosphorylation only of BCKDH E1␣ but not the highly homologous E1␣ su...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Roles of Amino Acid Residues Surrounding Phosphorylation Site 1 of Branched-chain α-Ketoacid Dehydrogenase (BCKDH) in Catalysis and Phosphorylation Site Recognition by BCKDH Kinase

Hawes

Schnepf

Jenkins

et al. 1995

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The ␣-keto acid of leucine, KIC, is thought to inhibit the BCKD kinase, promoting activation of BCKD complex. BCKD kinase phosphorylates the E1␣ subunit of BCKD at two sites, termed sites 1 and 2 (26,51). Site-directed mutagenesis studies (66) have shown that phosphorylation at site 1 [Ser 293 (S293)] inhibits BCKD activity, whereas phosphorylation at site 2 is silent.…”

mentioning

confidence: 99%

Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR

Lynch

Halle²,

Fujii³

et al. 2003

American Journal of Physiology-Endocrinology and Metabolism

103

View full text Add to dashboard Cite

Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyper-plastic growth, and tissue morphogenesis in in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian target of rapamycin (mTOR), the cytosolic Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate-limiting step in leucine oxidation is catalyzed by the branched-chain α-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1α-subunit at Ser293 (S293), which inactivates the complex. Because the α-keto acid of leucine regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to mTOR activation, a phosphopeptide-specific antibody recognizing this site was developed and characterized. Phospho-S293 (pS293) immunoreactivity in liver corresponded closely to diet-induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylations in adipose tissue and gastrocnemius (which is mostly inactive in vivo) were similar. This suggests that BCKD complex in epididymal adipose tissue from food-deprived rats is mostly inactive (unable to oxidize leucine), as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pS293 antibodies. Increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (Thr389, the phosphorylation site leading to activation) in adipose tissue was maximal at a dose of leucine that increased plasma leucine approximately threefold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation than in the activation of the leucine signal to mTOR.

show abstract

“…A computer homology search of translated nucleotide and peptide sequence databases by using the deduced amino acid sequence of the S. avermitilis ORF1 and ORF2 sequences showed the best scores with E1␣ and E1␤ subunits of several BCDH and PDH complexes from different species (Table 1). A multiple amino acid sequence alignment of the S. avermitilis ORF1 product with those of the E1␣ BCDH and E1␣ PDH subunits in Table 1 showed similarity to structural motifs found in all E1␣ subunits of ␣-keto acid dehydrogenase complexes examined so far, such as the TPPbinding motif (25), a putative subunit interaction site (65), and the phosphorylation sites (I and II) of the E1␣ chains of the mammalian BCDH and PDH complexes (12,47,70) (Fig. 2).…”

Section: Resultsmentioning

confidence: 80%

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli

et al. 1995

View full text Add to dashboard Cite

A cluster of genes encoding the E1␣, E1␤, and E2 subunits of branched-chain ␣-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced. Open reading frame 1 (ORF1) (E1␣), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids). ORF2 (E1␤), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids). The intergenic distance between ORF1 and ORF2 is 73 bp. The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2. Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (␣ or ␤) of several prokaryotic and eukaryotic BCDH complexes. When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S. avermitilis ORF1 and ORF2 products, respectively, were detected. In addition, specific E1[␣␤] BCDH activity was detected in E. coli cells carrying the S. avermitilis ORF1 (E1␣) and ORF2 (E1␤) coexpressed under the control of the T7 promoter.The branched-chain ␣-keto acid dehydrogenase (BCDH) complex catalyzes the oxidative decarboxylations of ␣-ketoisovalerate, ␣-keto-␤-methylvalerate, and ␣-ketoisocaproate (the deamination products of the branched-chain amino acids valine, isoleucine, and leucine, respectively), releasing CO 2 and generating the corresponding acyl-coenzyme A and NADH (36). The enzyme has been characterized from several sources, including Pseudomonas putida (55), Pseudomonas aeruginosa (39), Bacillus subtilis (37), rabbit liver (46), and bovine and rat kidneys (43, 49). The purified complexes from P. putida, P. aeruginosa, B. subtilis, and several mammals are all composed of four polypeptides, E1␣, E1␤, E2, and E3, with three different catalytic activities: a BCDH and decarboxylase (E1[␣␤]), a dihydrolipoamide acyltransferase (E2), and a dihydrolipoamide dehydrogenase (E3). The E1[␣␤] component requires thiamine pyrophosphate (TPP) as a cofactor and possesses a structural binding motif that resembles those of many of the TPP-binding enzymes (25). The BCDH complex has structural and enzymatic properties similar to those of pyruvate dehydrogenase (PDH) and ␣-ketoglutarate dehydrogenase complexes (48, 69). Interestingly, a dual-purpose ␣-keto acid dehydrogenase complex which has both pyruvate and BCDH activities has been isolated from B. subtilis (37). In addition, an exclusive BCDH which is essential for branchedchain fatty acid synthesis has been isolated from B. subtilis (44).Cloning of prokaryotic BCDH genes has been reported for Pseudomonas and Bacillus species but not for Streptomyces species. In these systems, it was found that the genes encoding the BCDH complex were clustered in an operon. The genes encoding the BCDH complex of P. putida (bkd genes) and the PDH/BCDH dual complex of B. subtilis and Bacillus stearothermophilus are clustered in the sequence E1␣, E1␤, E2, and E3 (10,11,26,27,59). Recently, the genes for the branchedchain fatty acid-specific BCDH fr...

show abstract

Phosphorylation sites and inactivation of branched-chain α-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle

Cited by 68 publications

References 24 publications

Roles of Amino Acid Residues Surrounding Phosphorylation Site 1 of Branched-chain α-Ketoacid Dehydrogenase (BCKDH) in Catalysis and Phosphorylation Site Recognition by BCKDH Kinase

Roles of Amino Acid Residues Surrounding Phosphorylation Site 1 of Branched-chain α-Ketoacid Dehydrogenase (BCKDH) in Catalysis and Phosphorylation Site Recognition by BCKDH Kinase

Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli

Contact Info

Product

Resources

About