title: Yeast pyruvate decarboxylase tetramers can dissociate into dimers along two interfaces. Hybrids of low-activity D28A (or D28N) and E477Q variants, with substitution of adjacent active center acidic groups from different subunits, display restored activity.
abstract: The tetrameric enzyme yeast pyruvate decarboxylase (YPDC) has been known to dissociate into dimers at elevated pH values. However, the interface along which the dissociation occurs, as well as the fundamental kinetic properties of the resulting dimers, remains unknown. The active sites of YPDC are comprised of amino acid residues from two subunits, a property which we utilize to address the issue as to which dimer interface is cleaved under different conditions of dissociation. Hydroxide-induced dissociation of the active site D28A (or D28N) and E477Q variants, each at least 100 times less reactive than wild-type YPDC, followed by reassociation of D28A (or D28N) and E477Q variants led to a remarkable 35-50-fold increase in activity. This result is possible only if the hydroxide-induced dissociation results in a cleavage along the interface between two subunits so that residues D28 and E477 are now separated. Upon reassociation, one of the two active sites of the hybrid dimer will have both residues substituted, whereas the second one will be of the wild-type phenotype. In contrast to the hydroxide-induced dimers, the urea-induced dissociation recently proposed results in dissociation along dimer-dimer interfaces, without separating the active sites, and therefore, on reassociation, these dimers do not regain activity. The significance of the results is discussed in light of a recently proposed alternating sites mechanism for YPDC. A preparative ion-exchange method is reported for the separation and purification of hybrid enzymes.
authors: Department of Chemistry and Program in Cellular and Molecular Biodynamics, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA esergienko@traidt.com.
authors: Eduard A
authors: EA
authors: Sergienko
authors: Frank
authors: F
authors: Jordan
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D019281:Dimerization; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D021122:Protein Subunits; D011767:Pyruvate Decarboxylase