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Title: Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain. cap alpha. -ketoacid dehydrogenase (BCKDH)

Abstract

BCKDH is phosphorylated by a copurifying kinase at two serine residues on the El..cap alpha.. subunit. Phosphorylation of both sites occurs at about the same rate initially, but inactivation is believed associated only with site 1 phosphorylation. The effects of KPi and known inhibitors of BCKDH kinase, ..cap alpha..-chloroisocaproate (CIC) and branched chain ..cap alpha..-ketoacids (BCKA), on the phosphorylation of purified rat liver BCKDH were studied. Site-specific phosphorylation was quantitated by thin-layer electrophoresis of tryptic peptides followed by densitometric scanning of autoradiograms. Addition of 5 mM KPi was found necessary to stabilize the BCKDH activity at 37/sup 0/C. Increasing the KPi to 50 mM dramatically increased the CIC and BCKA inhibition of site 1 and site 2 phosphorylation. The finding of enhanced sensitivity of inhibitors with 50 mM KPi may facilitate identification of physiologically important kinase effectors. Regardless of the KPi concentration, CIC and the BCKA showed much more effective inhibition of site 2 than site 1 phosphorylation. Although site 1 is the primary inactivating site, predominant inhibition of site 2 phosphorylation may provide a means of modulating kinase/phosphatase control of BCKDH activity under steady state conditions.

Authors:
; ; ;
Publication Date:
Research Org.:
Indiana Univ. School of Medicine, Indianapolis
OSTI Identifier:
6179702
Report Number(s):
CONF-870644-
Journal ID: CODEN: FEPRA; TRN: 87-034025
Resource Type:
Conference
Resource Relation:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States); Journal Volume: 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ENZYME INHIBITORS; BIOCHEMICAL REACTION KINETICS; OXIDOREDUCTASES; ENZYME ACTIVITY; PHOSPHORYLATION; AUTORADIOGRAPHY; ELECTROPHORESIS; PHOSPHATES; POTASSIUM COMPOUNDS; STEADY-STATE CONDITIONS; ALKALI METAL COMPOUNDS; CHEMICAL REACTIONS; ENZYMES; KINETICS; OXYGEN COMPOUNDS; PHOSPHORUS COMPOUNDS; REACTION KINETICS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Kuntz, M.J., Shimomura, Y., Ozawa, T., and Harris, R.A.. Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain. cap alpha. -ketoacid dehydrogenase (BCKDH). United States: N. p., 1987. Web.
Kuntz, M.J., Shimomura, Y., Ozawa, T., & Harris, R.A.. Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain. cap alpha. -ketoacid dehydrogenase (BCKDH). United States.
Kuntz, M.J., Shimomura, Y., Ozawa, T., and Harris, R.A.. 1987. "Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain. cap alpha. -ketoacid dehydrogenase (BCKDH)". United States. doi:.
@article{osti_6179702,
title = {Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain. cap alpha. -ketoacid dehydrogenase (BCKDH)},
author = {Kuntz, M.J. and Shimomura, Y. and Ozawa, T. and Harris, R.A.},
abstractNote = {BCKDH is phosphorylated by a copurifying kinase at two serine residues on the El..cap alpha.. subunit. Phosphorylation of both sites occurs at about the same rate initially, but inactivation is believed associated only with site 1 phosphorylation. The effects of KPi and known inhibitors of BCKDH kinase, ..cap alpha..-chloroisocaproate (CIC) and branched chain ..cap alpha..-ketoacids (BCKA), on the phosphorylation of purified rat liver BCKDH were studied. Site-specific phosphorylation was quantitated by thin-layer electrophoresis of tryptic peptides followed by densitometric scanning of autoradiograms. Addition of 5 mM KPi was found necessary to stabilize the BCKDH activity at 37/sup 0/C. Increasing the KPi to 50 mM dramatically increased the CIC and BCKA inhibition of site 1 and site 2 phosphorylation. The finding of enhanced sensitivity of inhibitors with 50 mM KPi may facilitate identification of physiologically important kinase effectors. Regardless of the KPi concentration, CIC and the BCKA showed much more effective inhibition of site 2 than site 1 phosphorylation. Although site 1 is the primary inactivating site, predominant inhibition of site 2 phosphorylation may provide a means of modulating kinase/phosphatase control of BCKDH activity under steady state conditions.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 46:6,
place = {United States},
year = 1987,
month = 5
}

Conference:
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  • Polyclonal antibodies (anti-E1E2 IgG) directed against bovine kidney BCKD have been used to examine the metabolic role of this enzyme in the rat. BCKD activity was assayed in detergent-disrupted kidney mitochondria using (1-/sup 14/C)..cap alpha..-ketoacids. Rates of oxidation of the keto analogs of leucine, valine and isoleucine were 21.6 +/- 1.5, 20.6 +/- 1.3 and 10.2 +/- 0.6 nmol/min/mg protein, respectively. Addition of anti-E1E2 IgG completely inhibited oxidation of all 3 ketoacids. Anti-E1E2 IgG inhibited oxidation of the keto analogs derived from methionine and threonine by 75% and 30%, respectively. It did not inhibit mitochondrial dehydrogenases other than BCKD. Thus,more » BCKD appears to be important in oxidative metabolism of 5 of the 9 indispensable amino acids. Immunoblots of rat kidneys, liver, muscle and heart mitochondria revealed a tissue specific alteration in the E1..cap alpha.. subunit of BCKD. Kidneys and heart each appeared to contain two E1..cap alpha.. polypeptides differing by an apparent molecular weight of 900 daltons; the predominant E1..cap alpha.. polypeptide in heart was the heavier E1..cap alpha.. band whereas in kidney it was the lighter band. Liver and muscle, however, each exhibited a single but different E1..cap alpha.. polypeptide. E1..cap alpha.. in liver corresponded to the lighter E1..cap alpha.. polypeptide of kidney and heart whereas in muscle E1..cap alpha.. corresponded to the heavier polypeptide. The E1..cap alpha.. subunit differences are associated with differences in basal BCKD activity of these tissues.« less
  • The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearlymore » 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-{angstrom} resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other {alpha}-ketoacid dehydrogenase complexes.« less