title: Substitution studies of the second divalent metal cation requirement of protein tyrosine kinase CSK.
abstract: In addition to a magnesium ion needed to form the ATP-Mg complex, we have previously determined that at least one more free Mg2+ ion is essential for the activation of the protein tyrosine kinase, Csk [Sun, G., and Budde, R. J. A. (1997) Biochemistry 36, 2139-2146]. In this paper, we report that several divalent metal cations, such as Mn2+, Co2+, Ni2+, and Zn2+ bind to the second Mg2+-binding site of Csk with up to 13200-fold higher affinity than Mg2+. This finding enabled us to substitute the free Mg2+ at this site with Mn2+, Co2+, Ni2+, or Zn2+ while keeping ATP saturated with Mg2+ to study the role of the free metal cation in Csk catalysis. Substitution by these divalent metal cations resulted in varied levels of Csk activity, with Mn2+ even more effective than Mg2+. Co2+ and Ni2+ supports reduced levels of Csk activity compared to Mg2+. Zn2+ has the highest affinity for the second Mg2+-binding site of Csk at 0.65 microM, but supports no kinase activity, acting as a dead-end inhibitor. The inhibition by Zn2+ is reversible and competitive against free Mg2+, noncompetitive against ATP-Mg, and mixed against the phosphate accepting substrate, polyE4Y, significantly increasing the affinity for this substrate. Substitution of the free Mg2+ with Mn2+, Co2+, or Ni2+ also results in lower Km values for the peptide substrate. These results suggest that the divalent metal activator is an important element in determining the affinity between Csk and the phosphate-accepting substrate.
authors.affiliation: Department of Neuro-Oncology, University of Texas, M. D. Anderson Cancer Center, Houston 77030, USA.
authors.forename: G
authors.initials: G
authors.lastname: Sun
authors.forename: R J
authors.initials: RJ
authors.lastname: Budde
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D001667:Binding, Competitive; D000081247:CSK Tyrosine-Protein Kinase; D002104:Cadmium / Q000737:chemistry / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent; D003035:Cobalt / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
chemical_list: D002413:Cations, Divalent; D008670:Metals; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D009532:Nickel; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D008274:Magnesium; D015032:Zinc