The Subsites Structure of Bovine Pancreatic Ribonuclease A Accounts for the Abnormal Kinetic Behavior with Cytidine 2′,3′-Cyclic Phosphate

Abstract

The kinetics of the hydrolysis of cytidine 2,3-cyclic phosphate (C>p) to 3-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p 2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p 2 binding subsite are used. 2) The k cat /K m values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the k cat obtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.Bovine pancreatic RNase A (EC 3.1.27.5) catalyzes the breakdown of RNA by means of a transesterification from the 5Ј position of one nucleotide to the 2Ј position of the adjacent nucleotide with the formation of a 2Ј,3Ј-cyclic phosphate product (Scheme 1). In an aqueous environment, the cyclic nucleotide is irreversibly hydrolyzed to a 3Ј nucleotide. The forward direction of the reaction requires a dinucleoside monophosphate as the minimum size for the substrate, whereas the reverse direction of the reaction can take place with pyrimidine 2Ј,3Ј-cyclic mononucleotides. It appears that the hydrolytic step is just a special case of the reverse transphosphorylation, as has been emphasized by Cuchillo et al. (1,2) and by Thompson et al. (3).Several kinetic studies with RNase A have been carried out following the hydrolysis of either uridine or cytidine 2Ј,3Ј-cyclic phosphate as substrates. At relatively low concentrations of this type of substrate, in an aqueous environment, the only significant route is that of hydrolysis, and a hyperbolic substrate concentration dependence is usually obtained. However, working at high substrate concentrations, Walker et al. (4,5) observed that at pH 7.0, the dependence of the rate of hydrolysis with substrate concentration shows a nonhyperbolic behavior: there is a drop at about 25 mM to 30 mM CϾp 1 followed by a second rise and then a gradual decline, which they interpreted by an allosteric model in which there is a substra...

Kinetic and Product Distribution Analysis of Human Eosinophil Cationic Protein Indicates a Subsite Arrangement That Favors Exonuclease-type Activity

“…We have studied in detail the substrate specificity and polynucleotide substrate cleavage pattern of this recombinant protein, and the results have been analyzed by ECP molecular modeling based on the three-dimensional structure of EDN and RNase A-substrate analog complexes. The distinct putative subsites configuration of ECP favors an exonuclease mechanism in the same way as the endonuclease activity of RNase A is a result of its own multisubsite structure 2 (22,23).…”

“…25 l of 5 mg/ml substrate solution were incubated with 5 l of 0.35 M RNase A, or 5 l of 7 M ECP for poly(U), and 5 l of 28 M ECP for poly(C) digestion. At different time intervals, between 0 and 90 min, the products of the reaction were analyzed as described previously (22). 15 l of the reaction mixture were injected on a reversed-phase HPLC column, NovaPak C 18 (size 3.9 ϫ 150 mm), equilibrated with 10% ammonium acetate (w/v) and 1% acetonitrile (v/v) in water, and after a 10-min wash, a 50-min linear gradient was applied from 0 to 90% of elution buffer (10% ammonium acetate (w/v) and 11% acetonitrile (v/v) in water).…”

“…4 and Ref. 22). In the case of RNase A, with a described endonuclease mechanism, there is a clear accumulation of intermediate products.…”

“…4). For comparison with the RNase A degradation of poly(C), the results of Moussaoui et al (22) were considered. The elution profile of the digestion products shows that with ECP the cyclic mononucleotide appears early in the incubation, especially with poly(C) as substrate, whereas most of the high molecular mass substrate still remains uncleaved.…”

Dehydrogenases from All Three Domains of Life Cleave RNA

“…The Dehydrogenases Cleave RNA between Pyrimidine and Adenine-RNase A is a single strand-specific RNase that preferentially cleaves 3Ј to pyrimidine residues (27). To determine the cleavage specificity of rabbit muscle GAPDH and the Asd-1 and Acd-5 proteins, primer extension analysis was performed (Fig.…”