title: Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum.
abstract: Pyruvate kinase activity is an important element in the flux control of the intermediate metabolism. The purified enzyme from Corynebacterium glutamicum demonstrated a marked sigmoidal dependence of the initial rate on the phosphoenolpyruvate concentration. In the presence of the negative allosteric effector ATP, the phosphoenolpyruvate concentration at the half-maximum rate (S0.5) increased from 1.2 to 2.8 mM, and cooperation, as expressed by the Hill coefficient, increased from 2.0 to 3.2. AMP promoted opposite effects: the S0.5 was decreased to 0.4 mM, and the enzyme exhibited almost no cooperation. The maximum reaction rate was 702 U/mg, which corresponded to an apparent kcat of 2,540 s-1. The enzyme was not influenced by fructose-1,6-diphosphate and used Mn2+ or Co2+ as cations. Sequence determination of the C. glutamicum pyk gene revealed an open reading frame coding for a polypeptide of 475 amino acids. From this information and the molecular mass of the native protein, it follows that the pyruvate kinase is a tetramer of 236 kDa. Comparison of the deduced polypeptide sequence with the sequences of other bacterial pyruvate kinases showed 39 to 44% homology, with some regions being very strongly conserved.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: M S
authors.initials: MS
authors.lastname: Jetten
authors.forename: M E
authors.initials: ME
authors.lastname: Gubler
authors.forename: S H
authors.initials: SH
authors.lastname: Lee
authors.forename: A J
authors.initials: AJ
authors.lastname: Sinskey
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D011770:Pyruvate Kinase