Freedom of assembly: metabolic enzymes come together

Abstract

Many different enzymes in intermediate metabolism dynamically assemble filamentous polymers in cells, often in response to changes in physiological conditions. Most of the enzyme filaments known to date have only been observed in cells, but in a handful of cases structural and biochemical studies have revealed the mechanisms and consequences of assembly. In general, enzyme polymerization functions as a mechanism to allosterically tune enzyme kinetics, and it may play a physiological role in integrating metabolic signaling. Here, we highlight some principles of metabolic filaments by focusing on two well-studied examples in nucleotide biosynthesis pathways-inosine-5'-monophosphate (IMP) dehydrogenase and cytosine triphosphate (CTP) synthase.

FIGURE 1:

A single amino acid change in the IMPDH N-terminal extension disrupts assembly of IMPDH octamers. Intermolecular interactions between IMPDH protomers are amplified eghtfold through reciprocal interactions across the polymerization interface between octamers. The avidity effects of such multivalent interactions may facilitate evolution of polymers of oligomeric proteins. Conversely, mutation of just one amino acid, tyrosine 12 to alanine, in the N-terminal extension, completely prevents octamer polymerization.

FIGURE 2:

Structures of CTPS and IMPDH filaments. (A) CTPS structures. CTPS monomers each have two catalytic domains (blue and green), and assemble into tetramers. Tetramers can dynamically assemble filaments with different geometries. A unique eukaryotic insert mediates assembly contacts of human CTPS, while Escherichia coli CTPS tetramers form more extensive, interlocking interactions. (B) Cellular polymerization of CTPS is broadly conserved (adapted from Barry et al. 2014; Gou et al., 2014). (C) IMPDH structures. IMPDH is an octamer, with each protomer consisting of catalytic (green) and regulatory (pink) domains. ATP binding in the regulatory domains stabilizes octamers, and GTP binding promotes a conformational change in that, in the context of the octamer and in the filament, results in a compressed, inactive conformation. (D) IMPDH polymers are observed to form in mouse lymphocytes upon TCR stimulation (adapted from Duong-Ly et al. 2018). (E) Cryo-tomography of HEp-2 cells shows IMPDH forming extensive bundles with spacing consistent with the spacing observed in in vitro reconstituted single filaments (adapted from Juda et al. 2014). Scale bars: B (top) = 10 μm, B (bottom) = 3 μm, D = 5 μm, E = 200 nm, inset in E = 10 nm.

FIGURE 3:

Potential effects of polymerization on enzyme kinetics. Hypothetical activity of free enzymes (blue) is compared with possible activity in filaments (green and orange). (A) Filaments can affect the catalytic rate constant, either increasing or decreasing activity across all concentrations of substrate. (B) Filaments might affect affinity for substrate (the apparent K_m), resulting in different activity levels at low substrate concentrations. (C) Filaments can influence the cooperativity of reactions. Circles show the 20 and 80% activity levels, highlighting the steepening of the activity curve with increasing cooperativity. (D) Filaments can shift the sensitivity to allosteric effectors, here illustrated as an inhibitor.