Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.
Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.
Biocrystallography Laboratory, Veterans Affairs Medical Center, P.O. Box 12055, University Drive C, Pittsburgh, Pennsylvania 15240, USA.
Palaniappa
P
Arjunan
Krishnamoorthy
K
Chandrasekhar
Martin
M
Sax
Andrew
A
Brunskill
Natalia
N
Nemeria
Frank
F
Jordan
William
W
Furey
D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D011485:Protein Binding; D011487:Protein Conformation; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; C014222:thiamine thiazolone pyrophosphate; D030481:Pyruvate Dehydrogenase (Lipoamide); C111305:pyruvate dehydrogenase E1 beta subunit; C065640:pyruvate dehydrogenase E1alpha subunit; D013835:Thiamine Pyrophosphate