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.
Highlights
The mitochondrial ␣-keto acid dehydrogenase multienzyme complexes, comprising the pyruvate dehydrogenase complex (PDC), the ␣-ketoglutarate dehydrogenase complex, and the branched-chain ␣-keto acid dehydrogenase (BCKDC), catalyze the oxidative decarboxylation of ␣-keto acids (Reaction 1) for energy production through the Krebs cycle [12, 13]
The mutation appears to lead to a small structural change in the phosphorylation loop, which alters the intermolecular interactions in this region and results in a new packing arrangement in the crystals, which is different from that reported previously for human E1b (16, 19 –22)
This work was prompted by the recent study with B. stearothermophilus E1p, which suggests the presence of proton wires to mediate communication between the two active sites [17]
Summary
MDa in size, are organized around a transacylase (E2) core, to which multiple copies of a decarboxylase (E1), a dehydrogenase (E3), and in the case of mammalian species a specific kinase and specific phosphatase are attached through noncovalent interactions. Chemical nonequivalence in the two active sites of the E1p component of human PDC has recently been demonstrated in the forms of ThDP C2 ionization rate, the ability to bind a substrate analog, and the decarboxylation rate constant of the 2-lactyl-ThDP intermediate [18] These results are consistent with the half-of-the-sites reactivity but shed no light on whether the proton-wire mechanism described for the B. stearothermophilus E1p [17] is responsible for the observed chemical nonequivalence between the two active sites in human E1p. The crystal structure of the E1b component from Thermus thermophilus BCKDC showed similar occupancy of the ␣-carbanion-ThDP intermediate in both active sites [23] These crystallographic properties of both human and Thermus E1bs are inconsistent with the half-of-the-sites model for these ThDP-dependent enzymes. We suggest that the inactive BCKDC observed with the phosphorylated E1b results from the specific geometry associated with the BCKDC assembly
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
