Role of divalent metals in the kinetic mechanism of insulin receptor tyrosine kinase.
The kinetic and thermodynamic interrelationships of peptide substrate (Val5-angiotensin 11), metal-ATP, and divalent metal cations with rat liver insulin receptor tyrosine kinase (IRTK) were investigated. Results of the initial rate studies with varying peptide and MnATP substrates indicates that the kinetic mechanism for IRTK is of the sequential type and therefore rules out a ping pong Bi Bi pathway. Hence, peptide substrate and metal-ATP bind to the kinase prior to the release of products. MnADP was a linear competitive inhibitor of MnATP and a noncompetitive inhibitor of peptide substrate. A synthetic tyrosine-containing pentapeptide, Glu-Glu-Phe-Tyr-Phe (EEFYF), was a linear competitive inhibitor of peptide substrate and a noncompetitive inhibitor of MnATP. Accordingly, the data show that phosphorylation of peptide substrate occurs via a rapid random equilibrium Bi Bi mechanism in which the kinase has the potential to react initially with either of the two substrates. In contrast, divalent metal cations and metal-ATP were found to interact with the kinase in a mutually inclusive manner, with metal binding to the kinase prior to MnATP. It was also found that divalent metals increase the affinity of the kinase for metal-ATP but do not affect the affinity of IRTK for metal-ADP product. Hence, divalent metals, during the reaction of association of enzyme with one of its substrates to form the binary complex, increase the relative concentration of E-ATP complex versus E-peptide complex, thus introducing a thermodynamic-dependent ordering for the interaction of substrates with the enzyme. To investigate the thermodynamics of this system, we assumed that under initial conditions the kinetic data we obtained reflected the association constants of reactants with the enzyme.
Vicario
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Rutgers State University, Department of Biological Sciences, Newark, New Jersey 07102.
Saperstein
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Bennun
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent; D004789:Enzyme Activation / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008670:Metals / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D013816:Thermodynamics
D002413:Cations, Divalent; D007328:Insulin; D008670:Metals; D010455:Peptides; D000804:Angiotensin II; C031193:angiotensin II, Asp(1)-Val(5)-; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin