title: Effects of metal ions, thiamine diphosphate analogues and subunit interactions on the reconstitution behaviour of pyruvate decarboxylase from brewer's yeast.
abstract: The reconstitution of pyruvate decarboxylase starts with reversible binding of thiamine diphosphate and Mg2(+)-ions to the apoenzyme, followed by a rate-limiting conformational change to the catalytically active holoenzyme. Investigations with diphospho-esters of 4-methyl-5-(2-hydroxyethyl)thiazolium derivatives have shown that the diphosphate residue of thiamine diphosphate is the most important part of the coenzyme responsible for the first reversible binding step. Methylation of the N1'-atom of the pyrimidine ring of thiamine diphosphate or 4'-oxythiamine diphosphate prevents the coenzyme from binding stably to the apoenzyme, so that the methylated coenzyme displays no coenzyme activity. In contrast, thiamine diphosphate analogues with bulky residues on the neighbouring C2'-atom of the pyrimidine ring form active holoenzyme complexes. This result shows the essential role of the N1'-atom of thiamine diphosphate in stable cofactor binding. The cofactor binding rate to the dimeric and tetrameric apoenzymes indicates that the cofactor is located in the contact regions of the subunits in the tetrameric enzyme.
authors.affiliation: Fachbereich Biochemie/Biotechnologie, Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg.
authors.forename: S
authors.initials: S
authors.lastname: Eppendorfer
authors.forename: S
authors.initials: S
authors.lastname: König
authors.forename: R
authors.initials: R
authors.lastname: Golbik
authors.forename: H
authors.initials: H
authors.lastname: Neef
authors.forename: K
authors.initials: K
authors.lastname: Lehle
authors.forename: R
authors.initials: R
authors.lastname: Jaenicke
authors.forename: A
authors.initials: A
authors.lastname: Schellenberger
authors.forename: G
authors.initials: G
authors.lastname: Hübner
mesh_terms: D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D046911:Macromolecular Substances; D008670:Metals / Q000378:metabolism*; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: D003067:Coenzymes; D046911:Macromolecular Substances; D008670:Metals; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate