Studies on tyrosine residues in porcine muscle adenylate kinase. Circular dichroism spectra and chemical modification with tetranitromethane.
Substrate-induced conformational change of porcine muscle adenylate kinase (EC 2.7.4.3) is evidenced by a change in circular dichroism spectra in the near ultraviolet. In the absence of tryptophan in porcine muscle adenylate kinase, the spectral change may be assigned to a perturbation of tyrosine chromophore(s). The spectral change was specific for adenine nucleotide binding and was greater with ATP than with AMP. In the x-ray model, Tyr153 and Tyr154 are located at a hinge region of two domains which form a deep active site cleft and are therefore susceptible to conformational change on substrate binding. Adenylate kinase was treated with equimolar tetranitromethane. The yellow-colored product, separated from unmodified enzyme by substrate gradient elution on a phosphocellulose column, had about 1 mol of nitrotyrosine per mol of the enzyme by amino acid analysis and showed a slightly higher Km value than native enzyme for ADP (Km = 0.50 mM compared with 0.25 mM for native adenylate kinase). Spectrophotometric titration of nitroadenylate kinase gave pKa 8.4 for the dissociation constant of the nitrotyrosyl hydroxyl group. On binding ATP the pKa value increased to 9.0 while AMP binding caused very little change. By peptide mapping of the carboxypeptidase digestion product, 0.70 mol of nitro group per mol of adenylate kinase was detected on Tyr153 and a small amount of nitro group was also found on Tyr95. From these results it is proposed that Tyr153 is directly or indirectly involved in the binding of ATP.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology*; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine; D013774:Tetranitromethane; D014443:Tyrosine
D000596:Amino Acids; D000249:Adenosine Monophosphate; D014443:Tyrosine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase; D008274:Magnesium; D013774:Tetranitromethane