[Spectroscopic study of the structure and intramolecular mobility of yeast pyruvate decarboxylase].
Steady-state and time-resolved fluorimetry were used to study the properties of holo- and apopyruvate decarboxylase (EC 4.1.1.1, PDC) from Brewer's yeast after interaction with substrate (pyruvate), cofactor (thiamine diphosphate, ThDP) and Mg2+ ions. The analysis of the enzyme's intrinsic fluorescence as well as of its complex with the probe 2-(p-toluidinylnaphthalene)-6-sulphonate (TNS) revealed that ThDP was found at the polar region of the PDC active sites, inducing a decrease in the mobility of the protein's nearest surroundings. The fluorescent probe had three different sites of binding to the protein apoform, two of which being located at the catalytic site and having different rotation freedom. The study of the PDC complex with thiochrome pyrophosphate, a ThDP structural analogue, pointed to the occurrence of a non-polar region of the enzyme active site for pyruvate absorption besides the polar region. The binding of pyruvate to the protein does not depend upon the cofactor's binding. On the basis of the fluorescent studies a model of the ThDP and pyruvate arrangement at the PDC active site is suggested.
Maskevich
S A
SA
Maskevich
A A
AA
Kivach
L N
LN
Chernikevich
I P
IP
Zabrodskaia
S V
SV
Oparin
D A
DA
D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D005456:Fluorescent Dyes; D008163:Luminescent Measurements; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013439:Sulfhydryl Reagents; D013831:Thiamine / Q000031:analogs & derivatives; D013835:Thiamine Pyrophosphate / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011773:Pyruvates; D013439:Sulfhydryl Reagents; C009460:thiochrome diphosphate; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D019289:Pyruvic Acid; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate; D013831:Thiamine
Spektroskopicheskie issledovaniia struktury i vnutrimolekuliarnoĭ podvizhnosti drozhzhevoĭ piruvatdekarboksilazy.