Hysteresis in human UDP-glucose dehydrogenase is due to a restrained hexameric structure that favors feedback inhibition.

Human UDP-α-d-glucose-6-dehydrogenase (hUGDH) displays hysteresis because of a slow isomerization from an inactive state (E*) to an active state (E). Here we show that the structure of E* constrains hUGDH in a conformation that favors feedback inhibition at physiological pH. The feedback inhibitor UDP-α-d-xylose (UDP-Xyl) competes with the substrate UDP-α-d-glucose for the active site. Upon binding, UDP-Xyl triggers an allosteric switch that changes the structure and affinity of the intersubunit interface to form a stable but inactive horseshoe-shaped hexamer. Using sedimentation velocity studies and a new crystal structure, we show that E* represents a stable conformational intermediate between the active and feedback-inhibited conformations. Because the allosteric switch occludes the cofactor and substrate binding sites in the inactive hexamer, the intermediate conformation observed in the crystal structure is consistent with the E* transient observed in relaxation studies. Steady-state analysis shows that the E* conformation enhances the affinity of hUGDH for the allosteric inhibitor UDP-Xyl by 8.6-fold (Ki = 810 nM). We present a model in which the constrained quaternary structure permits a small effector molecule to leverage a disproportionately large allosteric response.

Mediator redefines itself.

Mediator is a large and structurally dynamic protein complex that is globally required for eukaryotic transcription by RNA polymerase II. In a recent paper published in Cell Research, Wang et al. report for the first time the location of distinct subunits and redefine domains in the S. cerevisiae Mediator complex.

Hysteresis and negative cooperativity in human UDP-glucose dehydrogenase.

Human UDP-α-d-glucose 6-dehydrogenase (hUGDH) forms a hexamer that catalyzes the NAD(+)-dependent oxidation of UDP-α-d-glucose (UDG) to produce UDP-α-d-glucuronic acid. Mammalian UGDH displays hysteresis (observed as a lag in progress curves), indicating that the enzyme undergoes a slow transition from an inactive to an active state. Here we show that hUGDH is sensitive to product inhibition during the lag. The inhibition results in a systematic decrease in steady-state velocity and makes the lag appear to have a second-order dependence on enzyme concentration. Using transient-state kinetics, we confirm that the lag is in fact due to a substrate and cofactor-induced isomerization of the enzyme. We also show that the cofactor binds to the hUGDH:UDG complex with negative cooperativity. This suggests that the isomerization may be related to the formation of an asymmetric enzyme complex. We propose that the hysteresis in hUGDH is the consequence of a functional adaptation; by slowing the response of hUGDH to sudden increases in the flux of UDG, the other biochemical pathways that use this important metabolite (i.e., glycolysis) will have a competitive edge.

Cofactor binding triggers a molecular switch to allosterically activate human UDP-α-D-glucose 6-dehydrogenase.

Human UDP-α-D-glucose dehydrogenase (hUGDH) catalyzes the NAD(+)-dependent oxidation of UDP-α-D-glucose (UDG) to produce UDP-α-D-glucuronic acid. The oligomeric structure of hUGDH is dynamic and can form two distinct hexameric complexes in solution. The active form of hUGDH consists of dimers that undergo a concentration-dependent association to form a hexamer with 32 symmetry. In the presence of the allosteric feedback inhibitor UDP-α-D-xylose (UDX), hUGDH changes shape to form an inactive, horseshoe-shaped complex. Previous studies have identified the UDX-induced allosteric mechanism that changes the hexameric structure to inhibit the enzyme. Here, we investigate the role of the 32 symmetry hexamer in the catalytic cycle. We engineered a stable hUGDH dimer by introducing a charge-switch substitution (K94E) in the hexamer-building interface (hUGDH(K94E)). The k(cat) of hUGDH(K94E) is ~160-fold lower than that of the wild-type enzyme, suggesting that the hexamer is the catalytically relevant state. We also show that cofactor binding triggers the formation of the 32 symmetry hexamer, but UDG is needed for the stability of the complex. The hUGDH(K94E) crystal structure at 2.08 Å resolution identifies loop(88-110) as the cofactor-responsive allosteric switch that drives hexamer formation; loop(88-110) directly links cofactor binding to the stability of the hexamer-building interface. In the interface, loop(88-110) packs against the Thr131-loop/α6 helix, the allosteric switch that responds to the feedback inhibitor UDX. We also identify a structural element (the S-loop) that explains the indirect stabilization of the hexamer by substrate and supports a sequential, ordered binding of the substrate and cofactor. These observations support a model in which (i) UDG binds to the dimer and stabilizes the S-loop to promote cofactor binding and (ii) cofactor binding orders loop(88-110) to induce formation of the catalytically active hexamer.

Conformational flexibility in the allosteric regulation of human UDP-α-D-glucose 6-dehydrogenase.

UDP-α-D-xylose (UDX) acts as a feedback inhibitor of human UDP-α-D-glucose 6-dehydrogenase (hUGDH) by activating an unusual allosteric switch, the Thr131 loop. UDX binding induces the Thr131 loop to translate ~5 Å through the protein core, changing packing interactions and rotating a helix (α6(136-144)) to favor the formation of an inactive hexameric complex. But how does to conformational change occur given the steric packing constraints of the protein core? To answer this question, we deleted Val132 from the Thr131 loop to approximate an intermediate state in the allosteric transition. The 2.3 Å resolution crystal structure of the deletion construct (Δ132) reveals an open conformation that relaxes steric constraints and facilitates repacking of the protein core. Sedimentation velocity studies show that the open conformation stabilizes the Δ132 construct as a hexamer with point group symmetry 32, similar to that of the active complex. In contrast, the UDX-inhibited enzyme forms a lower-symmetry, horseshoe-shaped hexameric complex. We show that the Δ132 and UDX-inhibited structures have similar hexamer-building interfaces, suggesting that the hinge-bending motion represents a path for the allosteric transition between the different hexameric states. On the basis of (i) main chain flexibility and (ii) a model of the conformational change, we propose that hinge bending can occur as a concerted motion between adjacent subunits in the high-symmetry hexamer. We combine these results in a structurally detailed model for allosteric feedback inhibition and substrate--product exchange during the catalytic cycle.

Role of packing defects in the evolution of allostery and induced fit in human UDP-glucose dehydrogenase.

Allosteric feedback inhibition is the mechanism by which metabolic end products regulate their own biosynthesis by binding to an upstream enzyme. Despite its importance in controlling metabolism, there are relatively few allosteric mechanisms understood in detail. This is because allostery does not have an identifiable structural motif, making the discovery of new allosteric enzymes a difficult process. The lack of a conserved motif implies that the evolution of each allosteric mechanism is unique. Here we describe an atypical allosteric mechanism in human UDP-α-d-glucose 6-dehydrogenase (hUGDH) based on an easily acquired and identifiable structural attribute: packing defects in the protein core. In contrast to classic allostery, the active and allosteric sites in hUGDH are present as a single, bifunctional site. Using two new crystal structures, we show that binding of the feedback inhibitor, UDP-α-d-xylose, elicits a distinct induced-fit response; a buried loop translates ∼4 Å along and rotates ∼180° about the main chain axis, requiring surrounding side chains to repack. This allosteric transition is facilitated by packing defects, which negate the steric conformational restraints normally imposed by the protein core. Sedimentation velocity studies show that this repacking favors the formation of an inactive hexameric complex with unusual symmetry. We present evidence that hUGDH and the unrelated enzyme dCTP deaminase have converged to very similar atypical allosteric mechanisms using the same adaptive strategy, the selection for packing defects. Thus, the selection for packing defects is a robust mechanism for the evolution of allostery and induced fit.