Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases.
An enzyme of molecular weight 32,000 comprising a single subunit has been isolated from whole cell extracts of the yeast Saccharomyces cerevisiae. In vitro, the enzyme transfers the gamma phosphate of ATP to a protein substrate, histone H4, to produce an alkali-stable phosphorylation. Modification of the substrate histidine with diethylpyrocarbonate prevented phosphorylation. Phosphoamino acid analysis of the phosphorylated substrate showed the presence of 1-phosphohistidine. Hence, the isolated enzyme is a protein histidine kinase. A novel assay for acid-labile alkali-stable protein phosphorylation was used in the purification of the kinase activity to a final specific activity of 2,700 nmol/15 min/mg. The purified enzyme phosphorylates specifically histidine 75 in histone H4 and does not phosphorylate histidine 18 nor histidine residues in any other core histone. Steady state kinetic data are consistent with an ordered sequential reaction with Km values for Mg-ATP and histone H4 of 60 and 17 microM, respectively. The protein histidine kinase requires a divalent cation such as Mg2+, Co2+, or Mn2+ but will not use Ca2+, Zn2+, Cu2+, Fe2+, spermine, or spermidine. This is the first purification of an enzyme that catalyzes N-linked phosphorylation in proteins.
Huang
J M
JM
Department of Biological Chemistry, University of California, Davis 95616.
Wei
Y F
YF
Kim
Y H
YH
Osterberg
L
L
Matthews
H R
HR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D000071677:Histidine Kinase; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D005656:Fungal Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase