In animal tissue, BCKDC catalyzes an irreversible step[2] in the catabolism of the branched-chain amino acids L-isoleucine, L-valine, and L-leucine, acting on their deaminated derivatives (L-alpha-keto-beta-methylvalerate, alpha-ketoisovalerate, and alpha-ketoisocaproate, respectively) and converting them[3] to α-Methylbutyryl-CoA, Isobutyryl-CoA and Isovaleryl-CoA respectively.[4][5][6] In bacteria, this enzyme participates in the synthesis of branched, long-chain fatty acids.[7] In plants, this enzyme is involved in the synthesis of branched, long-chain hydrocarbons.
The overall catabolic reaction catalyzed by the BCKDC is shown in Figure 1.
Figure 1: This is the overall reaction catalyzed by the branched-chain alpha-ketoacid dehydrogenase complex.
Structure
The mechanism of enzymatic catalysis by the BCKDC draws largely upon the elaborate structure of this large enzyme complex. This enzyme complex is composed of three catalytic components:
In humans, 24 copies of E2 arranged in octahedral symmetry form the core of the BCKDC.[8] Non-covalently linked to this polymer of 24 E2 subunits are 12 E1 α2β2 tetramers and 6 E3homodimers. In addition to the E1/E3-binding domain, there are 2 other important structural domains in the E2 subunit: (i) a lipoyl-bearing domain in the amino-terminal portion of the protein and (ii) an inner-core domain in the carboxy-terminal portion. The inner-core domain is linked to the other two domains of the E2 subunit by two interdomain segments (linkers).[9] The inner-core domain is necessary to form the oligomeric core of the enzyme complex and catalyzes the acyltransferase reaction (shown in the "Mechanism" section below).[10] The lipoyl domain of E2 is free to swing between the active sites of the E1, E2, and E3 subunits on the assembled BCKDC by virtue of the conformational flexibility of the aforementioned linkers (see Figure 2).[11][12] Thus, in terms of function as well as structure, the E2 component plays a central role in the overall reaction catalyzed by the BCKDC.
Figure 2:This is a schematic of the "swinging" lipoyl domain. Note that this lipoyl domain is covalently attached to the E2 subunit of the BCKDC, but is free to swing between the E1, E2, and E3 subunits. As is described in the "Mechanism" section, the ability of the lipoyl group to freely swing between the active sites on each of the three subunits of the BCKDC plays a large and important role in the catalytic activity of this enzyme complex.[13]
The role of each subunit is as follows:
E1 subunit
E1 uses thiamine pyrophosphate (TPP) as a catalytic cofactor. E1 catalyzes both the decarboxylation of the α-ketoacid and the subsequent reductive acylation of the lipoyl moiety (another catalytic cofactor) that is covalently bound to E2.
E2 subunit
E2 catalyzes a transfer of the acyl group from the lipoyl moiety to coenzyme A (a stoichiometric cofactor).[14]
E3 subunit
The E3 component is a flavoprotein, and it re-oxidizes the reduced lipoyl sulfur residues of E2 using FAD (a catalytic cofactor) as the oxidant. FAD then transfers these protons and electrons to NAD+ (a stoichiometric cofactor) to complete the reaction cycle.
Mechanism
As previously mentioned, BCKDC's primary function in mammals is to catalyze an irreversible step in the catabolism of branched-chain amino acids. However BCKDC has a relatively broad specificity, also oxidizing 4-methylthio-2-oxobutyrate and 2-oxobutyrate at comparable rates and with similar Km values as for its branched-chain amino acid substrates.[15] The BCKDC will also oxidize pyruvate, but at such a slow rate this side reaction has very little physiological significance.[16][17]
The reaction mechanism is as follows.[18] Please note that any of several branched-chain α-ketoacids could have been used as a starting material; for this example, α-ketoisovalerate was arbitrarily chosen as the BCKDC substrate.
NOTE: Steps 1 and 2 occur in the E1 domain
STEP 1: α-ketoisovalerate combines with TPP and is then decarboxylated. The proper arrow-pushing mechanism is shown in Figure 3.
Figure 3: α-ketoisovalerate combines with TPP and is then decarboxylated
STEP 2: The 2-methylpropanol-TPP is oxidized to form an acyl group while being simultaneously transferred to the lipoyl cofactor on E2. Note that TPP is regenerated. The proper arrow-pushing mechanism is shown in Figure 4.
Figure 4: The 2-methylpropanol-TPP is oxidized to form an acyl group while being simultaneously transferred to the lipoyl cofactor on E2. Note that TPP is regenerated.
NOTE: The acylated lipoyl arm now leaves E1 and swings into the E2 active site, where Step 3 occurs.
STEP 3: Acyl group transfer to CoA. The proper arrow-pushing mechanism is shown in Figure 5.
Figure 5: Acyl group transfer to CoA
*NOTE: The reduced lipoyl arm now swings into the E3 active site, where Steps 4 and 5 occur.
STEP 4: Oxidation of the lipoyl moiety by the FAD coenzyme, as shown in Figure 6.
Figure 6: Oxidation of the lipoyl moiety by the FAD coenzyme.
STEP 5: Reoxidation of FADH2 to FAD, producing NADH:
FADH2 + NAD+ → FAD + NADH + H+
Disease relevance
A deficiency in any of the enzymes of this complex as well as an inhibition of the complex as a whole leads to a buildup of branched-chain amino acids and their harmful derivatives in the body. These accumulations lend a sweet smell to bodily excretions (such as ear wax and urine), leading to a pathology known as maple syrup urine disease.[19]
Mutations of the BCKDK gene, whose protein product controls the activity of the complex, may result in over-activation of the complex and excessive catabolism of the three amino acids. This leads to branched-chain keto acid dehydrogenase kinase deficiency, a rare disease first described in humans in 2012.[21]
^Lennarz WJ; et al. (1961). "The role of isoleucine in the biosynthesis of branched-chain fatty acids by micrococcus lysodeikticus". Biochemical and Biophysical Research Communications. 6 (2): 1112–116. doi:10.1016/0006-291X(61)90395-3. PMID14463994.
^Chuang DT. (1989). "Molecular studies of mammalian branched-chain alpha-keto acid dehydrogenase complexes: domain structures, expression, and inborn errors". Annals of the New York Academy of Sciences. 573: 137–154. doi:10.1111/j.1749-6632.1989.tb14992.x. PMID2699394. S2CID33210101.
^Perham RN. (1991). "Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein". Biochemistry. 30 (35): 8501–8512. doi:10.1021/bi00099a001. PMID1888719.
^Berg, Jeremy M., John L. Tymoczko, Lubert Stryer, and Lubert Stryer. Biochemistry. 6th ed. New York: W.H. Freeman, 2007. 481. Print.
^Heffelfinger SC, Sewell ET, Danner DJ (1983). "Identification of specific subunits of highly purified bovine liver branched-chain ketoacid dehydrogenase". Biochemistry. 22 (24): 5519–5522. doi:10.1021/bi00293a011. PMID6652074.
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