Claudi M. Cuchillo

Bovine Pancreatic Ribonuclease: Fifty Years of the First Enzymatic Reaction Mechanism

Fifty years ago, the group of Tony Mathias and Bob Rabin at University College London deduced the first mechanism for catalysis by an enzyme, ribonuclease [Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., and Ross, C. A. (1961) Nature 190, 781-784]. Here, we celebrate this historic accomplishment by surveying knowledge of enzymology and protein science at that time, facts that led to the formulation of the mechanism, criticisms and alternative mechanisms, data that supported the proposed mechanism, and some of the refinements that have since provided a more precise picture of catalysis of RNA cleavage by ribonucleases. The Mathias and Rabin mechanism has appeared in numerous textbooks, monographs, and reviews and continues to have a profound impact on biochemistry.

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

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up) n U>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1sites, located upstream of the P-O-5′ cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.

The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane

Abstract.Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present.

9 – Pancreatic Ribonucleases

Publisher Summary This chapter reviews recent data on several aspects of the catalytic mechanism of pancreatic RNases as well as some molecular properties such as carbohydrate content and folding and unfolding pathways. This chapter begins with a discussion on reaction catalyzed by pancreatic ribonucleases. The depolymerization of RNA by RNase is described as taking place in two steps: in the first step there is a transphosphorylation reaction from the 5′ position of one nucleotide to the 2′ position of the adjacent nucleotide with the formation of a 2′,3′-cyclic phosphodiester. In the second step, the 2′,3′-cyclic phosphodiester is hydrolyzed to a 3′ nucleotide. The description of this reaction has been a source of ambiguity with respect to the mechanism, especially as to the role of the 2′,3′-cyclic phosphodiesters. This chapter explains concepts related to the specificity of reaction and catalytic mechanism. It also describes structure and functions of substrate-binding subsites. An overview of structure and function of carbohydrate moiety is presented. The chapter concludes with a discussion on folding/unfolding studies of reduced/native RNase A.

Surface-exposed amino acids of eosinophil cationic protein play a critical role in the inhibition of mammalian cell proliferation.

Eosinophil cationic protein (ECP) is a ribonuclease secreted from activated eosinophils that may cause tissue injure as a result of eosinophilic inflammation. ECP possesses bactericidal, antiviral and helminthotoxic activity and inhibits mammalian cell growth. The mechanism by which ECP exerts its toxicity is not known but it has been related to the ability of the protein to destabilise lipid bilayers. We have assessed the involvement of some cationic and aromatic surface exposed residues of ECP in the inhibition of proliferation of mammalian cell lines. We have constructed ECP mutants for the selected residues and assessed their ability to prevent cell growth. Trp10 and Trp35 together with the adjacent stacking residue are critical for the damaging effect of ECP on mammalian cell lines. These residues are also crucial for the membrane disruption activity of ECP. Other exposed aromatic residues packed against arginines (Arg75-Phe76 and Arg121-Tyr122) and specific cationic amino acids (Arg101and Arg104) of ECP play a secondary role in the cell growth inhibition. This may be related to the ability of the protein to bind carbohydrates such as those found on the surface of mammalian cells.

The severed activation segment of porcine pancreatic procarboxypeptidase a is a powerful inhibitor of the active enzyme Isolation and characterisation of the activation peptide

The activation peptide of the monomeric procarboxypeptidase A from porcine pancreas was isolated by means of controlled trypsin digestion of the proenzyme followed by ion-exchange chromatography under dissociating conditions (7 M urea). The molecular weight of the isolated peptide was estimated to be around 11500-12000 (corresponding to approx. 100-103 residues) as judged by SDS electrophoresis and amino acid analysis, a figure that agrees with the differences between the corresponding values for procarboxypeptidase A and carboxypeptidase A (peptidyl-L-amino acid hydrolase, EC 3.4.17.1). The activation peptide has a high content of hydrophobic and acidic amino acids, and lacks cysteine. A remarkable feature is the strong competitive inhibitory action of the peptide on both porcine and bovine pancreatic carboxypeptidase A activity, with a Ki in the nanomolar range, and its null ability to inhibit porcine pancreatic carboxypeptidase B (EC 3.4.17.2). The above properties, and the fact that the peptide has the same N-terminal residue (lysine) as the parent procarboxypeptidase A, suggest that the isolated peptide contains most (if not all) of the activation segment of the proenzyme.

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

Abstract. The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p1 ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p2 and p0 have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p2 , and Lys-66 in p0 ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p2 are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p0 electrostatic interaction contributes only to binding of the RNA.

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

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 p2 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 p2 binding subsite are used. 2) Thek 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 catobtained 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.

Urea-gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products

Monomeric procarboxypeptidase A (PCPA) and isolated proproteinase E (PPE), both from pig pancreas, were shown by means of electrophoresis on transverse urea gradients (0–9 M) to form a very stable complex, identical to their natural binary complex. Although the complex is maintained by the interaction of both active regions, the activation segment of PCPA participates directly in the binding. Procarboxypeptidase B (PCPB) also associates with PPE, but in this case the complex shows low stability. In contrast with carb‐oxypeptidases A that strongly bind to their corresponding severed activation segments, no interaction was observed between carboxypeptidase B and its severed activation segment. The above results give some insight into several characteristics of the structure and activation properties of pancreatic PCPA and PCPB.

Eosinophil-induced neurotoxicity: The role of eosinophil cationic protein/RNase 3

We analyze the effect of ECP on primary cultures of cerebellar granule cells (CGCs) and astrocytes in an effort to understand the role of ECP in the eosinophil-induced neurotoxicity. We have shown that ECP induces dose-dependent cell death in both CGCs and astrocytes. The effect of ECP action on cell morphology is consistent with apoptosis for both cell types. The apoptotic mechanism involves ECP binding on the cell surface and an increase in the free cytosolic Ca(2+) concentration. It is associated with the activation of caspase-3, -8 and -9, processes that are also involved in the apoptosis induced either by stroke or other neurodegenerative conditions. Our results open new insights to clarify the neurotoxic effects associated to ECP in the hypereosinophilic syndrome.