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

Abstract

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 exonucleaselike 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 (p 2 ) is lacking, the secondary base subsite (B 2 ) is severely impaired, and there are new interactions at the p o , B o , and p -1 sites, 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.

Topography Studies on the Membrane Interaction Mechanism of the Eosinophil Cationic Protein

“…WT ECP and W10K, W35A, and W35AR36A mutants were obtained from a human ECP synthetic gene (9). Mutants were constructed as described in ref 26.…”

“…Mutants were constructed as described in ref 26. Protein expression in the E. coli BL21(DE3) strain (Novagen, Madison, WI), folding of the proteins from inclusion bodies, and the purification steps were carried out as previously described (9).…”

A phosphate‐binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

“…In the case of the K7H/R10H/H12K/H119Q-RNase A variant a clear preference for the exonucleolytic versus endonucleolytic activity is seen (Fig. 6); the predicted three-dimensional structure of this variant does not show any binding region capable of establishing an electrostatic interaction with the substrate through the phosphate group located at the 39 side with respect to the phosphate that binds at the active site; this structural conformation may explain the similarity between the activity of this variant and the cleavage preference found with either K7Q/R10Q-RNase A or ECP (Boix et al 1999b;Cuchillo et al 2002). The behavior of the K7H/R10H-RNase A variant in what refers to the preference for an endo-or exonucleolytic activity is consistent with a small increase for the exoversus endonucleolytic activity with respect to the RNase A cleavage; the analysis of the cleavage pattern does not allow us to determine the specific contribution to the total reaction rate of each catalytic site in this variant (p 1 and p 2 ).…”

“…6; Cuchillo et al 2002). Studies of substrate cleavage with the eosinophil cationic protein (ECP) (also known as RNase 3) have also emphasized the relevance of this binding site for its endonucleolytic activity; this protein, a member of the RNase A protein family, shows a clear preference for the exonucleolytic cleavage of the substrate (Boix et al 1999b) at the same time that the X-ray three-dimensional structure indicates the absence of an equivalent substratebinding region (Boix et al 1999a). In the case of the K7H/R10H/H12K/H119Q-RNase A variant a clear preference for the exonucleolytic versus endonucleolytic activity is seen (Fig.…”

“…RNase A variants obtained by site-directed mutagenesis were purified according to the general procedure described by Boix et al (1999b). However, the steepness of the salt gradient applied to the cation exchange chromatography was adjusted to optimum separation in each case.…”