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J. Biol. Chem., Vol. 269, Issue 4, 2529-2534, Jan, 1994
E Boix, MV Nogues, CH Schein, SA Benner and CM Cuchillo
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 (Pares, X., Llorens, R.,
Arus, 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.
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
Departament de Bioquimica i Biologia Molecular (Faculty Ciencies), Universitat Autonoma de Barcelona, Bellaterra, Spain.
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