Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase

Beattie, Nathaniel R.; Keul, Nicholas D.; Sidlo, Andrew M.; Wood, Zachary A.

doi:10.1021/acs.biochem.6b01044

Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase

Journal Article · Wed Dec 14 00:00:00 EST 2016 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.6b01044· OSTI ID:1346223

Beattie, Nathaniel R. ^[1]; Keul, Nicholas D. ^[1]; Sidlo, Andrew M. ^[1]; ^[1]

Univ. of Georgia, Athens, GA (United States)

Human UDP-glucose dehydrogenase (hUGDH) is regulated by an atypical allosteric mechanism in which the feedback inhibitor UDP-xylose (UDP-Xyl) competes with the 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 (E^Ω). This allosteric transition buries residue A136 in the protein core to produce a subunit interface that favors the E^Ω structure. Here we use a methionine substitution to prevent the burial of A136 and trap the T131-loop/α6 switch in the active conformation. We show that hUGDH_A136M does not exhibit substrate cooperativity, which is strong evidence that the methionine substitution prevents the formation of the low-UDP-Glc-affinity E^Ω state. In addition, the inhibitor affinity of hUGDH_A136M is reduced 14-fold, which most likely represents the K_i for competitive inhibition in the absence of the allosteric transition to the higher-affinity E^Ω 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 hUGDH_A136M 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 hUGDH_A136M, which shows that the A136M substitution has stabilized the active conformation of the T131-loop/α6 allosteric switch. Furthermore, this work shows that the T131-loop/α6 allosteric switch couples allostery and hysteresis in hUGDH.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: National Inst. of Health

OSTI ID:: 1346223

Journal Information:: Biochemistry, Journal Name: Biochemistry Journal Issue: 1 Vol. 56; ISSN 0006-2960

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: ENGLISH

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59 BASIC BIOLOGICAL SCIENCES
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Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase

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