The Two Active Sites in Human Branched-chain α-Keto Acid Dehydrogenase Operate Independently without an Obligatory Alternating-site Mechanism

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 ␣-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 halfphosphorylated 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.Thiamine diphosphate (ThDP), 2 a derivative of thiamine or vitamin B 1 , is an essential cofactor for ThDP-dependent enzymes that mediate the decarboxylation of ␣-keto acids or the transfer of the glycoaldehyde moiety from a ketose to an aldose (1-3). ThDP-dependent enzymes in the form of homodimers (␣ 2 ), homotetramers (␣ 4 ), or heterotetramers (␣ 2 ␤ 2 ) contain ThDP-binding pockets that constitute the two or four active sites of these enzymes. A long standing controversy in the literature has been whether these multiple active sites in a ThDP-dependent enzyme communicate with each other to affect the enzymatic reaction. An alternating activesite mechanism, or half-of-the-sites reactivity, was proposed, based on the negative cooperativity of ThDP binding to apotransketolase (4 -7). However, the crystal structure of the ␣-carbanion of (␣,␤-dihydroxyethyl)thiamine diphosphate in the active sites of yeast transketolase shows that the intermediate is present in high occupancy in both active sites, which does not support the alternating active-site mechanism in this enzyme (8). Yeast pyruvate decarboxylase, an ␣ 4 -type ThDPdependent enzyme, was shown kinetically to have two distinct pairs of active sites that alternate during the decarboxylation reaction (9). This kinetic model is supported by th...

A Novel Branched-chain Amino Acid Metabolon

“…This mode of interaction would be facilitated by a symmetric model for E1, in which both active sites are equally functional and independent of each other. In support of the symmetric model, we have recently obtained evidence that both active sites in the E1 component of the human BCKD complex can operate independently without communication to the other active site (70). Each of the 12 copies of the E1 component is anchored to the individual E1/E3 binding domain of the E2 subunit that assembles into the 24-meric core scaffold of the BCKD complex.…”

Pyruvate Dehydrogenase Kinase-4 Structures Reveal a Metastable Open Conformation Fostering Robust Core-free Basal Activity

“…On the core, the PDK dimer and the E1p heterotetramer bind to the L2 domain and the adjacent downstream E1BD domain, respectively, on the E2p subunit. Therefore, only one of the two E1p active sites facing the kinase may be efficiently phosphorylated, resulting in half-site phosphorylation, analogous to the related E1b component of the branched-chain ␣-ketoacid dehydrogenase complex (64).…”

Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

“…It was suggested that the two active sites in this enzyme operate independently during catalysis (6). This enzyme, however, does not have an uninterrupted array of acidic residues between ThDPs.…”

“…Communication between the thiamin diphosphate cofactors (ThDPs) 4 at the active centers of ThDP enzymes has been reported for several years (1)(2)(3)(4)(5)(6)). An intriguing pathway for such interaction had been suggested by Perham's group (2) on the basis of crystal structure studies on E1bs (the E1 component of the pyruvate dehydrogenase complex (PDHc) from Bacillus stearothermophilus).…”