title: Pyruvate decarboxylase activity is regulated by the Ser/Thr protein phosphatase Sit4p in the yeast Saccharomyces cerevisiae.
abstract: Deletion of SIT4 phosphatase decreased the pyruvate decarboxylase activity, which is essential for directing the glucose flux to ethanol production. Concomitantly, a reduction in the fermentative capacity was observed. As pyruvate decarboxylase expression was not altered, its post-translational phosphorylation was studied. Immunoblot analyses using anti-phosphoserine antibodies against the affinity-purified Pdc1p showed that Pdc1p is a phosphoenzyme. Dephosphorylation of Pdc1p by alkaline phosphatase inhibited activity by 50%. Moreover, phosphorylation of Pdc1p was dependent on the growth phase, being hyperphosphorylated in the logarithmic phase, which showed to be dependent on the presence of SIT4. A comparison of the kinetic parameters of pyruvate decarboxylase in total protein extracts from WT yeast and the Δsit4 mutant revealed that the apparent K(m) values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 μM, respectively, with V(max) values of 0.294 and 0.173 μmol mg⁻¹ min⁻¹, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the K(m) for TPP from 20 to 84 μM and for pyruvate from 2.3 to 4.6 mM, while the V(max) changed from 0.806 to 0.673 μmol mg⁻¹ min⁻¹. These results suggest that the Pdc1p phosphorylation dependent on SIT4 occurs at residues that change the apparent affinity for TPP and pyruvate.
authors.affiliation: Instituto de Bioquímica Médica Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Leandro José
authors.initials: LJ
authors.lastname: de Assis
authors.forename: Russolina Benedeta
authors.initials: RB
authors.lastname: Zingali
authors.forename: Claudio Akio
authors.initials: CA
authors.lastname: Masuda
authors.forename: Silas Pessini
authors.initials: SP
authors.lastname: Rodrigues
authors.forename: Monica
authors.initials: M
authors.lastname: Montero-Lomelí
mesh_terms: D003067:Coenzymes / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000378:metabolism
chemical_list: D003067:Coenzymes; D029701:Saccharomyces cerevisiae Proteins; D019289:Pyruvic Acid; D054648:Protein Phosphatase 2; C104343:SIT4 protein, S cerevisiae; C101504:PDC1 protein, S cerevisiae; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate
keywords: fermentation; pyruvate decarboxylase; sit4