Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase

Abstract

Human UDP-glucose dehydrogenase (hUGDH) is regulated by an atypical allosteric mechanism in which the feedback inhibitor UDP-xylose (UDP-Xyl) competes with substrate for the active site. Binding of UDP-Xyl triggers the T131-Loop/α6 allosteric switch, which converts the hexameric structure of hUGDH into an inactive, horseshoe-shaped complex (EΩ). This allosteric transition buries residue A136 in the protein core to produce a subunit interface that favors the EΩ structure. Here we use a methionine substitution to prevent the burial of A136 and trap the T131-Loop/α6 in the active conformation. We show that hUGDHA136M does not exhibit substrate cooperativity, which is strong evidence that the methionine substitution prevents the formation of the low UDP-Glc affinity EΩ state. In addition, the inhibitor affinity of hUGDHA136M is reduced 14 fold, which most likely represents the Ki for competitive inhibition in the absence of the allosteric transition to the higher affinity EΩ state. hUGDH also displays a lag in progress curves, which is caused by a slow, substrate-induced isomerization that activates the enzyme. Stopped flow analysis shows that hUGDHA136M does not exhibit hysteresis, which suggests that the T131-Loop/α6 switch is the source of the slow isomerization. This interpretation is supported by the 2.05 Å resolution crystal structure of hUGDHA136M, which shows that the A136M substitution has stabilized the active conformation of the T131-loop/α6 allosteric switch. This work shows that the T131-Loop/α6 allosteric switch couples allostery and hysteresis in hUGDH.

Hysteresis and Allostery in Human UDP-Glucose Dehydrogenase Require a Flexible Protein Core

“…18, 19, 23 This hypothesis is supported by a previous study that showed trapping the allosteric switch in the active conformation abolishes hysteresis. 23 The remarkable plasticity exhibited by the hUGDH protein core likely originates from large cavities and deep surface pockets (declivities) near the allosteric switch (Figure 1D). 15 Here, we have examined how the flexibility of the hUGDH protein core contributes to both hysteresis and allostery.…”

“…The NAD + saturation curves of hUGDH A104L are hyperbolic (Hill=1), with a K M of 48±3 μM (Figure 5C, Table 3). In contrast, the NAD + saturation curves of native hUGDH are known to display negative cooperativity 18, 19, 23 (Figure 5D Table 3). Earlier work showed that UDP-Glc binding induces an asymmetry that results in a mixture of high and low affinity NAD + binding sites.…”

“…Attempts to crystallize hUGDH A104L in the E Ω state using both of the previously published conditions 15 were not successful. However, the allosteric transition from E Ω to the E conformation can be observed in steady state inhibition studies of native hUGDH 18, 23 . Briefly, UDP-Xyl binding favors the E Ω state, which has a low affinity for UDP-Glc.…”

“…Because native hUGDH is hysteretic, the steady state velocities were calculated by fitting progress curves to eq.2 as described above. 15, 18 – 21, 23 hUGDH A104L does not display hysteresis the steady state velocity was determined by fitting the initial liner portion of the progress curves to a straight line. Initial steady state velocities from progress curves were fit to a sigmoidal rate equation and analyzed with residual plots 38 : v=normal kcatnormal [Et][S]hKMh+normal [S]h where h is the Hill coefficient.…”

“…The negative cooperativity observed in hUGDH NAD + substrate saturation curves was evaluated using Kurganov’s analysis as described previously. 23, 39 Briefly, the data were fit to the equation: v[S]=Vmax−vKmeff, where Kmeff= K0+(Klim−K0)(v/Vmax) where K 0 is the approximate K M for the high affinity binding sites and K lim is the average apparent K M for the low affinity sites. 39…”