Allosteric Domain Structure and Function

The first X-ray crystal structure of RePC, co-crystallized with ethyl-CoA, revealed the existence of the allosteric domain, which is positioned between the BC and CT domains and at the base of the carrier domain (PDB ID: 2QF7) (Figure I-3) (St. Maurice, et al., 2007). The allosteric domain has a simple composition in PC, consisting of a central a-helix covered by four antiparallel b-strands partially wrapping around it. The a-helix and b-strands are non-contiguous, with the helix originating from the C- terminal end of the BC domain and the four antiparallel b-strands located C-terminal to the CT domain. In the symmetric tetrameric conformation captured in the SaPC structure, the allosteric domains of the top face of the tetramer formed a dimerization interface with the allosteric domains on the bottom face of the tetramer. Thus, this domain has also been named the PC tetramerization domain (Xiang and Tong, 2008). This domain has no strong sequence conservation in other biotin-dependent enzymes, though a very similar structural motif has been observed in several other biotin-dependent enzymes. The BT domain of acyl-CoA carboxylases is one example of a similarly structured domain that resembles the PC allosteric domain. However, rather than containing four b-strands like the PC allosteric domain, the BT domain has eight b-strands that wrap around the central a-helix (Tong, 2017)

Since the discovery and initial characterizations of PC, it was known that acyl- CoA derivatives activated PC (Scrutton, et al., 1965). Acetyl-CoA is commonly the most effective activator of PC and has been the most extensively studied, along with allosteric inhibitors such as L-aspartate. The first crystal structure of RePC showed ethyl-CoA (a

nonhydrolyzable analogue of acetyl-CoA) bound in the allosteric domain (Figure I-3). Acetyl-CoA is stabilized in the binding site by hydrogen bonding with two conserved arginine residues, Arg427 and Arg472 in RePC (St. Maurice, et al., 2007). Mutations to these residues have an impact beyond simply affecting acetyl-CoA binding. For example, the Km for ATP is increased by mutations to these residues. (Adina-Zada, et al., 2012).

Many site-specific mutations have been made in this domain to elucidate the mechanism of allosteric activation by acetyl-CoA.

Activation by Acetyl-CoA. Soon after PC was initially purified, it was discovered

that acetyl-CoA acted as an activator (Scrutton, et al., 1965). Acetyl-CoA is generated cellularly by the pyruvate dehydrogenase complex for entry into the TCA cycle or by fatty acid oxidation. As acetyl-CoA enters into the TCA cycle, it condenses with oxaloacetate, forming citrate. This is a logical feedback mechanism, in which a higher concentration of acetyl-CoA, perhaps derived from an increased flux through glycolysis, requires an increased concentration of oxaloacetate with which to condense.

Several kinetic studies have determined that the BC domain is the locus of action for acetyl-CoA. Acetyl-CoA was observed to increase the magnitude of the dissociation rate constant for fluorescent analogues of both ATP and ADP (Geeves, et al., 1995). One initial study suggested that acetyl-CoA accelerates the formation of the carboxyphosphate intermediate and ATP cleavage. ATP cleavage was also better coupled to carboxybiotin

formation in the presence of acetyl-CoA, as well (Legge, Branson, Attwood, 1996). This was supported by a more recent study in which chimeric PC enzymes constructed from two isoforms of yeast PC (PYC1 and PYC2) were investigated for their regulatory properties. Despite having different degrees and characteristics of activation by acetyl- CoA, it was determined that the BC domain was the locus of acetyl-CoA activation (Jitrapakdee, et al., 2007).

Several recent studies have suggested that the role of acetyl-CoA is not simply confined to the BC domain. A 2011 study by Zeczycki, et al. showed that, under ideal conditions, acetyl-CoA enhanced the coupling between the BC domain and CT domain reactions, reducing non-productive ATP cleavage (Zecycki, et al., 2011). Succeeding this study was a thermodynamic and kinetic study conducted in SaPC to investigate the activation of acetyl-CoA. Acetyl-CoA was proposed to not only constrain the movements of the carrier domain, but also to kinetically and thermodynamically couple MgATP and pyruvate. The authors suggested that acetyl-CoA may even regulate the quaternary conformation and domain motions of PC (Westerhold, et al., 2018). This is consistent with suggestions by Sirithanakorn, et al. that acetyl-CoA may stabilize the symmetric conformation in PC, which involves a conformational change in the quaternary structure and corresponds to a repositioning of the carrier domain (Sirithanakorn, et al., 2016). However, direct observations of the effects of acetyl-CoA on carrier domain positioning or conformational dynamics of PC in response to acetyl-CoA are currently lacking.

Inhibition by L-aspartate. As a product of the TCA cycle, L-aspartate displays

classic feedback inhibition of PC. Studies of L-aspartate inhibition have generally been

reduce the degree of activation by acetyl-CoA. However, studies of PC cloned from three different species revealed that acetyl-CoA and L-aspartate binding are mutually

exclusive, suggesting that they share a common binding site (Cazzulo and Stoppani, 1968; Osmani, et al., 1981; Sirithanakorn, et al., 2014). Supporting this, Osmani, et al. suggested the binding site to be outside of the BC active site, as its inhibition with respect to MgATP is non-competitive in Aspergillus nidulans PC whereas an observation of competitive inhibition would have suggested a shared binding site between MgATP and

L-aspartate (Osmani, et al., 1981). Despite being mutually exclusive, the two effectors

appear to have distinct binding sites: mutation of the acetyl-CoA binding site does not affect L-aspartate inhibition in RePC (Adina-Zada, et al., 2012). No crystal structure has yet revealed the L-aspartate binding site, preventing a complete understanding of how L-

aspartate competes with acetyl-CoA and inhibits BC domain activity.

The locus of action of L-aspartate is the BC domain and not the CT domain (Sirithanakorn, et al., 2014). L-aspartate does not affect the coupling between BC and CT domain reactions whereas acetyl-CoA greatly affects half-reaction coupling

(Sirithanakorn, et al., 2014; Zeczycki, et al., 2011B). Finally, L-aspartate was observed to

similarly affect all carrier domain translocation pathways in RePC while acetyl-CoA preferentially activated the translocation pathway between the intramolecular BC domain and intermolecular CT domain (Liu, et al., 2018). The mechanism by which L-aspartate

inhibits PC catalytic activity remains unresolved and direct observations of the effect of