Enzymatic complexes across scales

Abstract

An unprecedented opportunity to integrate ~100 years of meticulous in vitro biomolecular research is currently provided in the light of recent advances in methods to visualize closer-to-native architectures of biomolecular machines, and metabolic enzymes in particular. Traditional views of enzymes, namely biomolecular machines, only partially explain their role, organization and kinetics in the cellular milieu. Enzymes self- or hetero-associate, form fibers, may bind to membranes or cytoskeletal elements, have regulatory roles, associate into higher order assemblies (metabolons) or even actively participate in phase-separated membraneless organelles, and all the above in a transient, temporal and spatial manner in response to environmental changes or structural/functional changes of their assemblies. Here, we focus on traditional and emerging concepts in cellular biochemistry and discuss new opportunities in bridging structural, molecular and cellular analyses for metabolic pathways, accumulated over the years, highlighting functional aspects of enzymatic complexes discussed across different levels of spatial resolution.

Keywords: electron microscopy; enzymology; glycolysis; protein-protein interactions.

Figure 1. Levels of metabolic organization

(A) Catalytic domains accelerate one or few biochemical reactions; (B) enzymatic complexes may form homomultimers or heteromultimers, by organizing enzymatic domains to efficiently perform more complicated catalytic reactions. For example, by homomultimerization of enzymatic domains, enzyme complexes achieve efficient catalysis of higher concentrations of metabolites. Heteromultimerization may co-localize sequential metabolic enzymes of parts of metabolic pathways. (C) A complete metabolic pathway may be organized on scaffold molecules and its participating enzymes transiently interact to form an assembly of its individual components, therefore achieving regulation of the metabolic pathway as function of cellular needs. (D) Enzymatic complexes and metabolons may be confined in membrane-less or membrane-bound organelles, therefore, further increasing catalytic efficiency or localizing specialized metabolic processes (see text).

Figure 2. Substrate channeling in Tryptophan synthase

On the left, a cartoon representation of two catalytic domains of Tryptophan synthase; small molecules are also shown bound to the catalytic sites, which communicate via a hydrophobic tunnel shown with yellow dots. On the right, the mechanism of substrate channeling in Tryptophan synthase is shown from the α to the β subunit. Note that the molecule is a dimer of dimers (faded part on the right is the continuation of the symmetric dimer) and therefore, mechanistic complication is expected via, e.g. communication of allosteric sites.

Figure 3. Substrate channeling mechanisms in FAS

(A) Fungal FAS is composed of two chains, each of which includes four catalytic domains. These domains (explained in the Abbreviations list) are organized in a highly complex 2.6 MDa structure, and each is found six times in the structure, therefore forming 48-catalytic domain biomolecule, highly intricate in the organization of the catalytic domains. (B) A simplified mechanism of substrate shuttling in fungal FAS, where the acyl carrier protein (ACP) brings the newly synthesized fatty acid sequentially to each of the catalytic domains of FAS. Note that this wheel-like catalysis is present six times in the protein complex, and therefore, highly intricate allosteric communication is expected, which by now is completely unknown. (C) Single-particles from acquired cryo-electron micrographs ([2] and unpublished data) showing the direct interaction between FAS and various other protein complexes, possibly forming a metabolon in fatty acid metabolism. The biomolecules often show connecting densities, clearly illustrating the interconnectivity of large protein complex assemblies and their direct interaction.

Figure 4. The glycolytic metabolon

(A) Simplified schematic of glycolysis and acronyms of participating enzymes. (B) A hypothesized glycolytic metabolon, where enzymes are interacting, shuttling the intermediates from an active site to another, while enzymatic domains are scaffolded on F-actin. Such a structure represents a classical metabolon which incorporates a full metabolic pathway. (C) Localization of the glycolytic metabolon in cells. The glycolytic metabolon has been identified close to plasma membrane of erythrocytes, in specialized organelles in trypanosomes (glycosome), close to mitochondria and on actin or tubulin filaments or formed under cellular stress in yeast. In addition, co-localization of glycolytic enzymes has been identified in HeLa cells, and during e.g. cancer cell metastasis, relocalization has been observed of glycolytic enzymes to aid cell motility. Finally, the glycolytic metabolon has been found to be localized close to synapses in the brain as a cluster of glycolytic enzymes (for details see text).

Figure 5. Phase separation and connection to metabolism

(A) A phase diagram for any biomolecule showing various transitions of its state; a state of liquid–liquid phase separation can be achieved, theoretically for any biomolecule. However, the boundaries of transitions are different for each biomolecule. (B) Advantages of phase-separated biomolecules: their unique biophysical properties may regulate mesh size and chemical properties of the condensate, which enables condensates to serve as molecular filters, restricting the diffusion of molecules through their boundaries based on, e.g. their sizes. Phase-separated membraneless organelles can also act as storage deposits, for example of inactive enzymes that would otherwise be waste material (e.g. glyconeogenesis and glycolytic enzymes could in principle phase separate in such granules when one or another competing metabolic pathway is utilized). At the edge of the biological condensate, due to the viscosity and the surface tension of the liquid phase of the condensate, force is generated. Such force could in principle, be utilized by various pathways that might require additional energy to complete. Condensates also function as organizing centers for polymerization, as shown for example, for actin filaments or the microtubule-organizing centers; however, it is yet unknown if enzymatic filaments follow an analogous principle. Signal amplification is also an attractive idea, where for example, inactive metabolic pathways become active after phase-separated signaling molecules, which may reach critical concentrations to regulate the ‘firing’ of the enzymatic pathway. Metabolons may organize on membranes via liquid–liquid demixing and phase-separation processes, having an affinity toward the boundaries of membranes (see also text). (C) Illustration of the usual phosphorylation patterns of proteins in phase-separated membraneless organelles which have effect on phase transitions, e.g. by decreasing or increasing biomolecular binding affinity. (D) Regulators/effectors of phase transitions.