Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum.

TitleStructural and functional analysis of pyruvate kinase from Corynebacterium glutamicum.
Publication TypeJournal Article
Year of Publication1994
AuthorsJetten, MS, Gubler, ME, Lee, SH, Sinskey, AJ
JournalAppl Environ Microbiol
Volume60
Issue7
Pagination2501-7
Date Published1994 Jul
ISSN0099-2240
KeywordsAmino Acid Sequence, Base Sequence, Corynebacterium, DNA, Bacterial, Genes, Bacterial, Kinetics, Molecular Sequence Data, Molecular Structure, Molecular Weight, Protein Conformation, Pyruvate Kinase, Restriction Mapping, Sequence Homology, Amino Acid, Substrate Specificity
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.

DOI10.1128/aem.60.7.2501-2507.1994
Alternate JournalAppl Environ Microbiol
Citation Key123
PubMed ID8074528
PubMed Central IDPMC201676