Studies on adenosine triphosphate transphosphorylases XIV. Equilibrium binding properties of the crystalline rabbit and calf muscle ATP-AMP transphosphorylase (adenylate kinase) and derived peptide fragments☆

Abstract Both a fluorescence-quenching technique and a uv-difference spectral method have been used to study the binding of 1, N 6 -etheno analogs of the adenine nucleotides (ϵATP, ϵADP, ϵAMP) ( J. A. Secrist III, J. R. Barrio, N. J., Leonard, and G. Weber, 1972 , Biochemistry , 11 , 3499–3506) to crystalline rabbit and calf muscle ATP-AMP transphosphorylase in the presence and absence of Mg 2+ , at 0.16 ( Γ 2 ), 25 °C, and pH 7.4. In addition, the binding of the ϵ-analogs of the adenine nucleotides has been studied to two S -[ 14 C]carboxymethylated peptide fragments of the rabbit muscle enzyme (residues 1–44 = MT-I; residues 171–193 = MT-XII), as well as to a synthetic nonapeptide corresponding to residues 32 − 40 of the rabbit muscle enzyme. In the case of the rabbit and calf enzymes: MgϵATP 2− , ϵATP 4− , MgϵADP − , and ϵAMP 2− are bound stoichiometrically ( n ∼- 1), MgϵAMP is insignificantly bound, and n ∼- 2 for ϵADP 3− ( n = maximal number of moles bound per mole of protein). In the case of S -carboxymethylated peptide fragments: MT-I binds stoichiometrically to MgϵATP 2− , ϵATP 4− , MgϵADP − , and ϵADP 3− with values of n ∼- 1; but MT-I does not bind to ϵAMP 2− significantly. MT-XII binds stoichiometrically to uncomplexed ϵAMP 2− or to uncomplexed ϵADP 3− (both with n ∼- 1); whereas, the binding of MgϵADP − , ϵATP 4− , and MgϵAMP to MT-XII are comparatively insignificant. Other peptide fragments in the molecule, viz. fragments MT-IV (residues 77–96) or MT-VI (residues 106–126) did not bind significantly to any of the ethenoanalogs; nor did insulin, nor, e.g., did bo vine serum albumin. The binding of the etheno analogs was also studied to an equimolar mixture of peptides MT-I + MT-XII, which qualitatively duplicated the binding pattern of the entire native molecule, and except for ϵATP 4− or MgϵATP 2− (which are bound more tightly to the entire native molecule), even quantitatively. The synthetic peptide (residues 32 to 40) was found to bind to MgϵATP 2− , ϵATP 4− , and MgϵADP − , with n ∼- 1; but it does not significantly bind to ϵAMP 2− , nor to ϵADP 3− . These binding data support the idea that there are two separate sites for the binding of either (a) the complexed nucleotide substrate (MgATP 2− or MgADP − ) residing in the sequence of MT-I (residues 1 to 44) and in the neighborhood of residues 32 to 40, or (b) the uncomplexed nucleotide substrate (AMP 2− or ADP 3− ) residing in the sequence of MT-XII (residues 171 to 193) of the rabbit muscle enzyme.