| Autor: |
Beattie NR; Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States., Keul ND; Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States., Sidlo AM; Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States., Wood ZA; Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States. |
| Jazyk: |
angličtina |
| Zdroj: |
Biochemistry [Biochemistry] 2017 Jan 10; Vol. 56 (1), pp. 202-211. Date of Electronic Publication: 2016 Dec 22. |
| DOI: |
10.1021/acs.biochem.6b01044 |
| Abstrakt: |
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. This work shows that the T131-loop/α6 allosteric switch couples allostery and hysteresis in hUGDH. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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