Journal of Molecular Biology
ReviewMembrane Lipids Assist Catalysis by CTP: Phosphocholine Cytidylyltransferase
Graphical abstract
Section snippets
CCT Activity Is Regulated by Reversible Membrane Binding
CCT is the rate-limiting and regulatory enzyme in the Kennedy pathway for the synthesis of phosphatidylcholine (PC) (Figure 1(a)), the dominant pathway for de novo PC synthesis in nearly all eukaryotic cells [2]. Its substrates and products are water-soluble, and the CCT active site is accessible to solvent. However, CCT requires a means to respond to changing concentrations of the pathway end-product, PC. This is accomplished by a membrane–lipid sensor domain (domain M), linked to the
Regulation of the Equilibrium between Soluble and Membrane-Bound Forms
The membrane partitioning of CCT is regulated by the membrane lipid composition and by the phosphorylation status of the region C-terminal to domain M (Figure 1(b)). A potential third influencing factor is the available protein-free space for embedding into the membrane surface. The features of the membrane and the amphipathic helical M domain that are responsible for stable binding and the antagonism of binding by phosphorylation have been reviewed recently [3]. We provide an overview here,
The CCT-Catalyzed Reaction Is Driven by Electrostatic Forces
The CCT catalytic domain is a modified Rossman fold with a β-sheet scaffold of five parallel β strands flanked by six α-helices, and an active site at the base of the β sheet [74]. In metazoan CCTs, this fold is capped by an N-terminal extension of ~ 35 ordered residues that interact with the catalytic fold and stabilize the dimer [74]. The job of the enzyme, beyond capturing the substrates, is to promote a U-shape of CTP, convert the α-phosphate from tetrahedral to the trigonal-planar geometry
Inhibition Mechanism
The activity of purified CCTα is very low (~ 50 M−1 s−1) and rises to ≥ 10,000 in the presence of added lipid, typically added in the form of small sonicated vesicles containing acidic lipid. The change is a reflection of a ~ 10-fold drop in the Km for CTP, but no change in the Km for phosphocholine, and a 20- to 30-fold increase in kcat [10,11,20,89]. The catalytic silencing device is housed in the most conserved and hydrophobic region of the M domain–a 22mer segment corresponding to residues
Membrane binding redirects the conformational ensemble of the allosteric inter-domain linker
Domain M has dual roles in regulation. It is inhibitory in the soluble form (CCTsol), as described above, and activating in the membrane form (CCTmem). The key evidence for dual roles is that deletion of domain M or specifically the AI segment is only partially activating [20,90], which is incompatible with a strictly auto-inhibitory function. For optimal membrane binding and activation, both the leash and the AI segment must be present [11,20,91,92]. How does the membrane-bound domain M
Benefits of a Reaction on the Membrane Surface: Electrostatic Enhancement of Key Catalytic Residues
A general strategy for catalysis, especially for reactions like CCT that rely on electrostatic enhancement, is the sequestration of active sites from water upon substrate binding to create a low-dielectric medium for the reaction. The charged and polarizable groups that catalyze the reaction can then forge ion pairs or H-bonds with substrates without interference from water and potentially neutralizing counter-ions [[94], [95], [96]]. This is often brought about by the movement of mobile loops
Similarities between the Activation Mechanism of CCT and Other Membrane-Bound Enzymes
Does the plasticity of the allosteric linker in CCT reflect a mechanism for activation that is common to other membrane enzymes with soluble catalytic domains? The enzyme with the most similar membrane activation mechanism is another enzyme of phospholipid metabolism, neutral sphingmyelinase (nSMase2). nSMase2 is activated by PS and other anionic phospholipids [103], and uses a membrane-associated allosteric linker, the juxta-membrane (JX) segment, to tether the catalytic domain to an integral
Membrane Participation Is Intrinsic to the CCT-Catalyzed Reaction, But to What Extent?
Membrane lipids participate in the CCT reaction on several levels. Firstly, the PC-deficient membrane captures CCT by providing a surface for folding and insertion of the domain M amphipathic helix. Increased membrane surface charge likely attracts the positively charged leash intervening between the J segment and the AI, and may selectively recruit dephosphorylated species [1] (Figure 5(b)). The leash–membrane interaction may assist in the dissociation of the AI helix, leading to αE helix
Acknowledgments
I thank Dr. Svetla Taneva for a critique of the manuscript. I would like to thank several Research Associates for their many contributions over the years to the analysis of the lipid activation of CCT: Dr. Jaeyong Lee, Dr. Svetla Taneva, Dr. Joanne Johnson, and Ziwei Ding. The research cited from my lab was funded by grants from CIHR and NSERC.
Declaration of Interest: None.
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