5TJH

hUGDH A136M Substitution

Summary for 5TJH

• Entry DOI: 10.2210/pdb5tjh/pdb
• Descriptor: UDP-glucose 6-dehydrogenase, PYROPHOSPHATE 2-, CHLORIDE ION, ... (5 entities in total)
• Functional Keywords: dehydrogenase, oxidoreductase
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 6
• Total formula weight: 332441.33

Authors

Beattie, N.R.,Wood, Z.A. (deposition date: 2016-10-04, release date: 2016-11-02, Last modification date: 2023-10-04)

Primary citation

Beattie, N.R.,Keul, N.D.,Sidlo, A.M.,Wood, Z.A.
Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase.
Biochemistry, 56:202-211, 2017

PubMed 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 (E). This allosteric transition buries residue A136 in the protein core to produce a subunit interface that favors the E 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 does not exhibit substrate cooperativity, which is strong evidence that the methionine substitution prevents the formation of the low-UDP-Glc-affinity E state. In addition, the inhibitor affinity of hUGDH is reduced 14-fold, which most likely represents the K for competitive inhibition in the absence of the allosteric transition to the higher-affinity E 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 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, 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.

PubMed: 27966912
DOI: 10.1021/acs.biochem.6b01044

Experimental method

X-RAY DIFFRACTION (2.05 Å)