T. Vang et al., KINETIC-PROPERTIES OF THE C-TERMINAL SRC KINASE, P50(CSK), Biochimica et biophysica acta. Protein structure and molecular enzymology, 1384(2), 1998, pp. 285-293
Csk is an important regulator of tyrosine kinases of the Src family. I
n this paper, we have characterised the kinetics and catalytic propert
ies of a highly active and stable enzyme obtained in milligram amounts
by expressing the enzyme as a fusion protein with glutathione-S-trans
ferase (GST) in Escherichia coli. Using the synthetic polyamino acid p
oly(Glu,Tyr) as substrate, phosphotransferase activity was linear for
7-8 min with Mg2+ and 5 min with Mn2+. With Mg2+ and Mn-2+,Mn- respect
ively, K-m (ATP) was 56.9 +/- 6.2 and 5.4 +/- 0.6 mu M and V-max was 2
93 +/- 52 and 217 +/- 38 pmol phosphate transferred (mu g Csk)(-1) min
(-1). Optimal concentrations of Mg2+ and Mn2+ were 4-10 mM and 2-3 mM,
respectively, and higher concentrations of both cations were inhibito
ry. The Csk activity was highly sensitive to monovalent (Na+, K+) and
divalent (Ca2+) cations, the sensitivity being 2-5-fold higher with Mg
2+ than Mn2+. Physiological concentrations of Ca2+ (less than 10 mu M)
were without effect. Autophosphorylation of Csk was demonstrated in v
itro, but did not influence the catalytic activity. Addition of inorga
nic phosphate above 100 mu M strongly inhibited Csk catalytic activity
towards poly(Glu,Tyr) in the presence of Mn2+, but not in the presenc
e of Mg2+. Phosphorylation of a physiological substrate (Lck) and auto
phosphorylation of Csk was not inhibited by phosphate, indicating that
the phosphate-dependent inhibition of Csk activity was substrate spec
ific. (C) 1998 Elsevier Science B.V. All rights reserved.