Rapid purification and biochemical characterization of glucose kinase from Streptomyces peucetius var. caesius.
Glucose kinase catalyzes the ATP-dependent phosphorylation of glucose. Streptomyces peucetius var. caesius glucose kinase was purified 292-fold to homogeneity. The enzyme has cytosolic localization and is composed of four identical subunits, each of 31 kDa. The purified enzyme easily dissociates into dimers. However, in the presence of 100 mM glucose the enzyme maintains its tetrameric form. Maximum activity was found at 42 degrees C and pH 7.5. Isoelectric focusing of the enzyme showed a pl of 8.4. The N- and C-terminal amino acid sequences were MGLTIGVD and VYFAREPDPIM, respectively. The kinetic mechanism of S. peucetius var. caesius glucose kinase appears to be a rapid equilibrium ordered type, i.e., ordered addition of substrates to the enzyme, where the first substrate is d-glucose. The K(m) values for d-glucose and MgATP(2-) were 1.6 +/- 0.2 and 0.8 +/- 0.1 mM, respectively. Mg(2+) in excess of 10 mM inhibits enzyme activity.
Imriskova
I
I
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F., 04510, México.
Langley
E
E
Arreguín-Espinosa
R
R
Aguilar
G
G
Pardo
J P
JP
Sánchez
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits; D020539:Sequence Analysis, Protein; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature
D046911:Macromolecular Substances; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D005947:Glucose