title: 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 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 (EΩ). This allosteric transition buries residue A136 in the protein core to produce a subunit interface that favors the EΩstructure. 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 hUGDHA136Mdoes not exhibit substrate cooperativity, which is strong evidence that the methionine substitution prevents the formation of the low-UDP-Glc-affinity EΩstate. In addition, the inhibitor affinity of hUGDHA136Mis reduced 14-fold, which most likely represents the Kifor competitive inhibition in the absence of the allosteric transition to the higher-affinity EΩstate. 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 hUGDHA136Mdoes 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.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Nathaniel R
authors.initials: NR
authors.lastname: Beattie
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Keul
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Sidlo
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Zachary A
authors.identifier: http://orcid.org/0000-0002-7251-0822
authors.initials: ZA
authors.lastname: Wood
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000494:Allosteric Regulation; D001667:Binding, Competitive; D055162:Biocatalysis; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D011487:Protein Conformation; D055503:Protein Multimerization; D013379:Substrate Specificity; D013997:Time Factors; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014540:Uridine Diphosphate Xylose / Q000378:metabolism*
chemical_list: D014540:Uridine Diphosphate Xylose; D008715:Methionine; D014533:Uridine Diphosphate Glucose Dehydrogenase; D000409:Alanine