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NMR characterization of substrate induced changes in structure and dynamics of human phosphomevalonate kinase
Author(s) -
Olson Andrew L,
Yao Huili,
Cai Shen,
Herdendorf Tim,
Miziorko Henry M,
Sem Daniel S
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1012.14
Subject(s) - binding site , dissociation constant , chemical shift , chemistry , stereochemistry , conformational change , ligand (biochemistry) , plasma protein binding , titration , biophysics , biochemistry , biology , receptor
Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, involving the transfer of a phosphoryl group from ATP to mevalonate‐5‐phosphate (M5P), producing mevalonate‐5‐diphosphate. This is the sole pathway for biosynthesis of steroids and isoprenoids in mammals. Despite the importance of this pathway in human cardiovascular disease, the mechanism of this enzyme is not well characterized. Here we have titrated both ligands to the protein, first M5P to free PMK then ADP. Then, titration of ADP to free PMK followed by M5P, monitoring chemical shift changes upon addition of these ligands using 1 H‐ 15 N HSQC‐NMR. The dynamic psec‐nsec timescale changes that occur in PMK with all complexes were characterized with NMR relaxation experiments (S 2 ). Following chemical shifts during ligand titration, we found they all bind with similar dissociation constants: 6 +/− 3 μM for M5P binding to free PMK and 56 +/− 16 uM for subsequent ADP binding; 19 +/− 9 μM for ADP binding to free PMK and <20 μM for subsequent binding of M5P. Chemical shift changes mapped onto a PMK structural model indicate that upon addition of Mg‐ADP the majority of changes are clustered around the binding pocket of ADP, however the binding of M5P causes changes throughout the whole protein. The first ligand bound induces the major conformational change, as there are few chemical shift changes in the ternary complex for both situations.