Separate effects of Mg2+, MgATP, and ATP4- on the kinetic mechanism for insulin receptor tyrosine kinase.
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 microM. The apparent Ki for ATP4- increased from 0.20 to 136 microM and the Hill coefficient 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 cooperativity. 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 concentration 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 concentrations 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- inhibitor, 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 between Mg2+ and MgATP, but also for the mutually exclusive 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 receptor tyrosine kinase is discussed which may account, at least in part, for modulation of cooperativity and the metal-dependent increase in turnover (VM).
Vicario
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PP
Department of Biochemical Endocrinology, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065.
Bennun
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A
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology*; D008962:Models, Theoretical; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism
D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D008274:Magnesium