STUDIES ON ADENOSINE-TRIPHOSPHATE TRANSPHOSPHORYLASES .14. EQUILIBRIUM BINDING-PROPERTIES OF THE CRYSTALLINE RABBIT AND CALF MUSCLE ATP-AMP TRANSPHOSPHORYLASE (ADENYLATE KINASE) AND DERIVED PEPTIDE-FRAGMENTS

Both a fluorescence-quenching technique and a uv-difference spectral method have been used to study the binding of 1,N6-etheno analogs of the adenine nucleotides (ε{lunate}ATP, ε{lunate}ADP, ε{lunate}AMP) (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 Mg2+, at 0.16 ( Γ 2), 25 °C, and pH 7.4. In addition, the binding of the ε{lunate}-analogs of the adenine nucleotides has been studied to two S-[14C]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: Mgε{lunate}ATP2-, ε{lunate}ATP4-, Mgε{lunate}ADP-, and ε{lunate}AMP2- are bound stoichiometrically (n ∼- 1), Mgε{lunate}AMP is insignificantly bound, and n ∼- 2 for ε{lunate}ADP3- (n = maximal number of moles bound per mole of protein). In the case of S-carboxymethylated peptide fragments: MT-I binds stoichiometrically to Mgε{lunate}ATP2-, ε{lunate}ATP4-, Mgε{lunate}ADP-, and ε{lunate}ADP3- with values of n ∼- 1; but MT-I does not bind to ε{lunate}AMP2- significantly. MT-XII binds stoichiometrically to uncomplexed ε{lunate}AMP2- or to uncomplexed ε{lunate}ADP3- (both with n ∼- 1); whereas, the binding of Mgε{lunate}ADP-, ε{lunate}ATP4-, and Mgε{lunate}AMP 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 ε{lunate}ATP4- or Mgε{lunate}ATP2- (which are bound more tightly to the entire native molecule), even quantitatively. The synthetic peptide (residues 32 to 40) was found to bind to Mgε{lunate}ATP2-, ε{lunate}ATP4-, and Mgε{lunate}ADP-, with n ∼- 1; but it does not significantly bind to ε{lunate}AMP2-, nor to ε{lunate}ADP3-. These binding data support the idea that there are two separate sites for the binding of either (a) the complexed nucleotide substrate (MgATP2- 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 (AMP2- or ADP3-) residing in the sequence of MT-XII (residues 171 to 193) of the rabbit muscle enzyme. © 1979.