Reverse transphosphorylation by ribonuclease A needs an intact p2-binding site. Point mutations at Lys-7 and Arg-10 alter the catalytic properties of the enzyme.
Bovine pancreatic ribonuclease A interacts with RNA along multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate. This work gives additional strong support to the existence of the postulated phosphate-binding subsite p2 (Parés, X., Llorens, R., Arús, C., and Cuchillo, C. M. (1980) Eur. J. Biochem. 105, 571-579) and confirms the central role of Lys-7 and Arg-10 in establishing an electrostatic interaction with a phosphate group of the substrate. The effects of charge elimination by Lys-7-->Gln (K7Q) and/or Arg-10-->Gln (R10Q) substitutions in catalytic and ligand-binding properties of ribonuclease A have been studied. The values of Km for cytidine 2',3'-cyclic phosphate and cytidylyl-3',5'-adenosine are not altered but are significantly increased for poly(C). In all cases, kcat values are lower. Synthetic activity, i.e. the reversion of the transphosphorylation reaction, is reduced for K7Q and R10Q mutants and is practically abolished in the double mutant. Finally, the extent of the reaction of the mutants with 6-chloropurine-9-beta-D-ribofuranosyl 5'-monophosphate indicates that the phosphate ionic interaction in p2 is weakened. Thus, p2 modification alters both the catalytic efficiency and the extent of the processes in which an interaction of the phosphate group of the substrate or ligand with the p2-binding subsite is involved.
Boix
E
E
Departament de Bioquímica i Biologia Molecular (Faculty Ciències), Universitat Autònoma de Barcelona, Bellaterra, Spain.
Nogués
M V
MV
Schein
C H
CH
Benner
S A
SA
Cuchillo
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D001120:Arginine; D012259:Ribonuclease, Pancreatic; D008239:Lysine