Archives of Biochemistry and Biophysics,
278, (1990)
Separate effects of Mg2+, MgATP and ATP4- on the
kinetic
mechanism for insulin receptor tyrosine kinase
Vicario, P.P. and Bennun, A.
The separate effects of the equilibrium species Mg2+, MgATP
substrate, and ATP4- on the reaction catalyzed by insulin
receptor tyrosine kinase were examined. The separated kinetic
constants show that the K0.5 value for Mg2+ decreased from
23 to 0.43 mM and the Hill coefficient for Mg2+ (hMg2+) decreased
from 1.43 to 0.668 when the concentration of ATPT (MgATP +
ATP4-) was increased from 50 to 1000 µM. The apparent
Ki for ATP4- increased from 0.20 to 136 µM and the Hill
co-efficient for ATP4- (hATP4-) decreased from 1.41 to 0.82
as the concentration of total ATP (ATPT) increased. These
findings suggest that the [ATP4-]/[Mg2+] ratio modulates the
shift from positive to negative coopera¬tivity. It was
also shown that the apparent affinity of the kinase for MgATP
increased as the concentration of free Mg2+ increased and
that the apparent affinity of the kinase for free Mg2+ increased
as the concentration of MgATP substrate increased. Thus, Mg2+
and MgATP interact with the kinase in a mutually inclusive
manner which leads to an increase in the ratio of the enzyme
(E) rate-limiting species, [Mg–E–MgATP]/[E–MgATP]. Free ATP4-
not only acts as a competitive inhibitor of the substrate
but also decreases the relative concentra¬tion of Mg–E–MgATP.
ATPT-dependent activation of the kinase is, therefore, a result
of MgATP's increasing the affinity of the kinase for Mg2+,
thereby leading to saturation of the enzyme with Mg2+ at lower
concentra¬tions of the divalent metal. This results in
an increase in the [Mg–E–MgATP]/[E–MgATP] ratio, and therefore
decreases saturation of the kinase with ATP4- inhibi¬tor,
not only at the active site but also at a kinetically distinct
regulatory site. This kinetic relationship allows not only
for the mutually inclusive interaction be¬tween Mg2+ and
MgATP, but also for the mutually ex¬clusive interaction
toward ATP4-, hence indicating that the effect of Mg2+ will
be to form an enzyme complex (Mg—E) which will have a higher
affinity for MgATP substrate and a lower affinity for ATP4-
than E alone. The role of the equilibrium concentrations of
Mg—E, E, and ATP—E on the activation of insulin recep¬tor
tyrosine kinase is discussed which may account, at least in
part, for modulation of cooperativity and the metal-dependent
increase in turnover (VM).