2J9F

Human branched-chain alpha-ketoacid dehydrogenase-decarboxylase E1b

Summary for 2J9F

• Entry DOI: 10.2210/pdb2j9f/pdb
• Related: 1DTW 1OLS 1OLU 1OLX 1U5B 1V11 1V16 1V1M 1V1R 1WCI 1X7W 1X7X 1X7Y 1X7Z 1X80 2BEU 2BEV 2BEW 2BFB 2BFC 2BFD 2BFE 2BFF
• Descriptor: 2-OXOISOVALERATE DEHYDROGENASE ALPHA SUBUNIT, 2-OXOISOVALERATE DEHYDROGENASE BETA SUBUNIT, MANGANESE (II) ION, ... (7 entities in total)
• Functional Keywords: oxidoreductase, flavoprotein, mitochondrion, thiamine pyrophosphate, maple syrup urine disease, transit peptide, phosphorylation, disease mutation, metal-binding, multi-enzyme complex
• Biological source: HOMO SAPIENS (HUMAN); More
• Total number of polymer chains: 4
• Total formula weight: 170388.12

Authors

Jun, L.,Machius, M.,Chuang, J.L.,Wynn, R.M.,Chuang, D.T. (deposition date: 2006-11-07, release date: 2007-02-27, Last modification date: 2023-12-13)

Primary citation

Li, J.,Machius, M.,Chuang, J.L.,Wynn, R.M.,Chuang, D.T.
The Two Active Sites in Human Branched-Chain Alpha- Keto Acid Dehydrogenase Operate Independently without an Obligatory Alternating-Site Mechanism.
J.Biol.Chem., 282:11904-, 2007

PubMed Abstract: A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction.

PubMed: 17329260
DOI: 10.1074/JBC.M610843200

Experimental method

X-RAY DIFFRACTION (1.88 Å)