Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum

Appl Environ Microbiol. 1994 Jul;60(7):2501-7. doi: 10.1128/aem.60.7.2501-2507.1994.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Corynebacterium / enzymology*
  • Corynebacterium / genetics
  • DNA, Bacterial / genetics
  • Genes, Bacterial
  • Kinetics
  • Molecular Sequence Data
  • Molecular Structure
  • Molecular Weight
  • Protein Conformation
  • Pyruvate Kinase / chemistry*
  • Pyruvate Kinase / genetics
  • Pyruvate Kinase / metabolism
  • Restriction Mapping
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • DNA, Bacterial
  • Pyruvate Kinase

Associated data

  • GENBANK/D13095
  • GENBANK/L07920
  • GENBANK/L27126
  • GENBANK/M24636
  • GENBANK/M63703