Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase

Biochemistry. 2017 Jan 10;56(1):202-211. doi: 10.1021/acs.biochem.6b01044. Epub 2016 Dec 22.

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 EΩ 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 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.

MeSH terms

  • Alanine / chemistry
  • Alanine / genetics
  • Alanine / metabolism
  • Allosteric Regulation*
  • Binding, Competitive
  • Biocatalysis
  • Catalytic Domain*
  • Crystallization
  • Crystallography, X-Ray
  • Humans
  • Kinetics
  • Methionine / chemistry
  • Methionine / genetics
  • Methionine / metabolism
  • Models, Molecular
  • Mutation, Missense
  • Protein Conformation
  • Protein Multimerization
  • Substrate Specificity
  • Time Factors
  • Uridine Diphosphate Glucose Dehydrogenase / chemistry
  • Uridine Diphosphate Glucose Dehydrogenase / genetics
  • Uridine Diphosphate Glucose Dehydrogenase / metabolism*
  • Uridine Diphosphate Xylose / metabolism*

Substances

  • Uridine Diphosphate Xylose
  • Methionine
  • Uridine Diphosphate Glucose Dehydrogenase
  • Alanine