Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR.

Abstract:

:The simultaneous observation of interdependent reactions within different phases as catalyzed by membrane-bound enzymes is still a challenging task. One such enzyme, the Escherichia coli integral membrane protein diacylglycerol kinase (DGK), is a key player in lipid regulation. It catalyzes the generation of phosphatidic acid within the membrane through the transfer of the γ-phosphate from soluble MgATP to membrane-bound diacylglycerol. We demonstrate that time-resolved (31)P magic angle spinning NMR offers a unique opportunity to simultaneously and directly detect both ATP hydrolysis and diacylglycerol phosphorylation. This experiment demonstrates that solid-state NMR provides a general approach for the kinetic analysis of coupled reactions at the membrane interface regardless of their compartmentalization. The enzymatic activity of DGK was probed with different lipid substrates as well as ATP analogs. Our data yield conclusions about intersubunit cooperativity, reaction stoichiometries and phosphoryl transfer mechanism and are discussed in the context of known structural data.

journal_name

Nat Chem Biol

journal_title

Nature chemical biology

authors

Ullrich SJ,Hellmich UA,Ullrich S,Glaubitz C

doi

10.1038/nchembio.543

subject

Has Abstract

pub_date

2011-05-01 00:00:00

pages

263-70

issue

5

eissn

1552-4450

issn

1552-4469

pii

nchembio.543

journal_volume

7

pub_type

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