PXD020676

PXD020676 is an original dataset announced via ProteomeXchange.

Title	The catalytic mechanism of vitamin K epoxide reduction in a cellular environment
Description	Vitamin K epoxide reductases (VKOR) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, how hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analysis to show that KO forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this covalent reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has a considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveal the catalytic mechanism of this integral membrane enzyme in a cellular environment.
HostingRepository	PRIDE
AnnounceDate	2020-12-03
AnnouncementXML	Submission_2020-12-02_23:14:28.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Guomin Shen
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	N-ethylmaleimide derivatized cysteine
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2020-07-31 08:24:11	ID requested
⏵ 1	2020-12-02 23:14:28	announced

Dataset with its publication pending

submitter keyword: LC/MS/MS,Human VKOR, catalytic mechanism

Weikai Li
contact affiliation	Washington University in Saint Louis
contact email	weikai@wustl.edu
lab head
Guomin Shen
contact affiliation	Henan University of Science and Technology
contact email	shenba433@163.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD020676
     1. Label: PRIDE project
     2. Name: The catalytic mechanism of vitamin K epoxide reduction in a cellular environment