Proton magnetic resonance spectra or porcine muscle adenylate kinase and substrate complexes.
Porcine muscle adenylate kinase with a molecular weight of 22,000 has 2 histidine, 5 phenylalanine, 7 tyrosine, and no tryptophan residues. The effect of pH, substrate, and the paramagnetic manganous ion on the proton magnetic resonance spectrum of the enzyme, particularly the aromatic region, has been investigated at 220 MHz. The well resolved C2 proton peaks of the 2 histidine residues have been individually assigned to His-36 and His-189 by comparison with the spectrum of the carp muscle enzyme which has only one C2 proton peak and only 1 histidine residue, 36. The chemical shift of the peak designated C2-H of His-36 in the porcine enzyme has a normal titration curve with a pKalpha = 6.3 but the peak for His-189 is not titratable in the pH range 5.8 to 8.1. The pKalpha of the single His-36 of the carp enzyme is similar to that of His-36 of the porcine enzyme. Changes in pH, particularly at low pH, also affect the chemical shifts of the tyrosine residues. Occupation of either the monophosphate site by AMP or the triphosphate site by ATP or GTP causes a downfield shift of the C2-H of His-36, and the equilibrium mixture causes an even greater shift, but no shift in the C2-H of His-189. The substrates also induce changes in the chemical shifts in the phenylalanine-tyrosine region of the spectrum. Tentative assignments of the highest and lowest field peaks in this region have been made based on the three-dimensional structure determined by x-ray crystallography. On the basis of these assignments, it is concluded that Phe-183 is unperturbed by substrate binding but that Tyr-153 or -154 at the hinge of the molecule, are perturbed. The C2-H of adenine and C8-H of adenine or guanine of the bound substrates were also observed; those of AMP are unperturbed but C2-H of ATO is shifted downfield and the C8-H of ATP and GTP are shifted upfield. The paramagnetic manganous ion had no effect on the spectrum at Mn(II) to enzyme ratios below 1:10; above this ratio, a general broadening was observed...
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002347:Carps; D006160:Guanosine Triphosphate; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese; D008961:Models, Structural; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010770:Phosphotransferases; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013045:Species Specificity; D013552:Swine
D000596:Amino Acids; D000249:Adenosine Monophosphate; D008345:Manganese; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase