Chemical modification by pyridoxal 5'-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A.
Steric and chemical evidence had previously shown that residues Lys-7 and/or Arg-10 of bovine pancreatic RNAase A could belong to the p2 phosphate-binding subsite, adjacent to the 3' side of the main site p1. In the present work chemical modification of the enzyme with pyridoxal 5'-phosphate and cyclohexane-1,2-dione was carried out in order to identify these residues positively as part of the p2 site. The reaction with pyridoxal 5'-phosphate yields three monosubstituted derivatives, at Lys-1, Lys-7 and Lys-41. A strong decrease in the yield of derivatives at Lys-7 and Lys-41 was observed when either p1 or p2 was specifically blocked by 5'-AMP or 3'-AMP respectively. These experiments indicate that both sites are needed for the reaction of pyridoxal 5'-phosphate with RNAase A to take place. The positive charge in one of the sites interacts with the phosphate group of pyridoxal 5'-phosphate, giving the proper orientation to the carbonyl group, which then reacts with the lysine residue present in the other site. The absence of reaction between pyridoxal 5'-phosphate and an RNAase derivative that has the p2 site blocked supports this hypothesis. Labelling of Lys-7 with pyridoxal 5'-phosphate has a more pronounced effect on the kinetics with RNA than with the smaller substrate 2',3'-cyclic CMP. In addition, when the phosphate moiety of the 5'-phosphopyridoxyl group was removed with alkaline phosphatase the kinetic constants with 2',3'-cyclic CMP returned to values very similar to those of the native enzyme, whereas a higher Km and lower Vmax. were still observed for RNA. This indicates that this new derivative has recovered a free p1 site and, hence, the capability to act on 2',3'-cyclic CMP, but the presence of the pyridoxyl group bound to Lys-7 is still blocking a secondary phosphate-binding site, namely p2. Finally, reaction of cyclohexane-1,2-dione at Arg-10 is suppressed in the presence of 3'-AMP but only a 19% decrease is observed with 5'-AMP, suggesting that Arg-10 is also close to the p2 phosphate-binding subsite.
Richardson
R M
RM
Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, Spain.
Parés
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Cuchillo
C M
CM
D000818:Animals; D001120:Arginine / Q000502:physiology; D001665:Binding Sites; D002417:Cattle; D003510:Cyclohexanes / Q000494:pharmacology*; D003512:Cyclohexanones / Q000494:pharmacology*; D008239:Lysine / Q000502:physiology; D010710:Phosphates / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D012259:Ribonuclease, Pancreatic / Q000032:analysis*
D003510:Cyclohexanes; D003512:Cyclohexanones; D010710:Phosphates; D011732:Pyridoxal Phosphate; C018031:1,2-cyclohexanedione; D001120:Arginine; D012259:Ribonuclease, Pancreatic; D008239:Lysine