Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates. Evidence for reversible tight-binding inhibition.
Variants of the pyruvate dehydrogenase subunit (E1; EC ) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. The K(m) values for thiamin diphosphate (ThDP) were 1.58 microm (parental E1) and 6.65 microm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 microm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr(177) substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K(i) = 0.003 microm for ThTDP and K(i) = 0.064 microm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr(177). Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr(177), the kinetic and spectroscopic data suggest that Tyr(177) has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.
Nemeria
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N
Department of Chemistry and the Program in Cellular and Molecular Biodynamics, Rutgers University, Newark, New Jersey 07102, USA.
Yan
Y
Y
Zhang
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Brown
A M
AM
Arjunan
P
P
Furey
W
W
Guest
J R
JR
Jordan
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013831:Thiamine / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D011768:Pyruvate Dehydrogenase Complex; D013844:Thiazoles; D014443:Tyrosine; D013831:Thiamine