Isomerization RNase

Vithayathil et al. (34%). In 0.5 M HC1 at 30° RNase-A undergoes structural alterations which can be detected chromatographically at neutral pH. However, all the products are equally active enzymically, and no reaction would have been detected by assay. At pH 11.0 even more involved structural changes take place quite rapidly. Irreversible alkaline denaturation takes place at higher pH and is very rapid at 13. Here the activity loss is accompanied by marked spectral changes indicating reactions such as / elimination at cystine or serine residues (343). A temperature-induced isomerization at neutral pH has been reported by French and Hammes. This is discussed in a later section on nucleotide binding. 

The probable general structure of the dimers was established in elegant experiments by Fruchter and Crestfield 381) involving alkylation with iodoacetate. The two isomeric dimers referred to above behave identically in these reactions. The two active sites in the dimers behave just like that of the monomer. Histidines 12 and 119 both react, but the reactions are mutually exclusive. The proposed structure is outlined in Fig. 19. The tail of one monomer combines with the body of the other and vice versa. The His 12 and 119 pairs are now on separate molecules. When the dimers, fully inactivated by reaction with iodoacetate, are dissociated by heating at neutral pH, the following monomers would be expected native RNase (active), CM-His-12-RNase (inactive) CM-His-119-RNase (inactive), and di-CM-His-12-His-119-RNase (inactive). These were, in fact, found. About 2b% activity reappeared from the inactive dimer. Equally important the di-CM compound was found. This material... 

Hammes (4S6) has summarized some of the extensive studies from his laboratory on the interaction of a variety of nucleotides with RNase-A as seen by relaxation kinetic measurements. The bimolecular and isomerization steps that occur with each of the nucleotides are very much faster than the rate determining steps separating the different substances. Thus the kinetic parameters for the interaction of each nucleotide can be established separately and then combined with steady state kinetic data to provide a detailed kinetic picture. The bimolecular steps are recognized by the concentration dependence of the relaxation time and the isomerization steps by the lack of a concentration dependence. 

Fig. 24. Kinetic mechanism for the interaction of substrates and products with RNase. The various bimolecular association steps and isomerization processes are shown. Proton binding and pH dependence are not indicated. [Adapted from Hararaes (466), Fig. 2 note that second isomerization originally inserted between EP2 and ESi was probably the result of a second binding site at high 3 -CMP concentration see Hammes and Walz (468).]...

Fid. 25. Kinetic mechanisms for nucleotide binding including proton ionization steps, (a) Proposed mechanism for the initial interaction of RNase with 3 -UMP. PH is the monoanionic nucleotide, and P is the dianionic nucleotide. The horizontal arrows represent the kinetically significant steps in the pH range 4.5-7.0 (free protons are not indicated on the diagram), (b) Proposed mechanism for the isomerization of enzyme 3 -UMP complexes. Reproduced from del Rosario and Hammes (470). 

The fraction of Us molecules depends on the number of proline residues and on their isomeric state in the native protein. In particular, the presence of cts-prolyl peptide bonds in the folded molecules leads to a high fraction of Us, since in unfolded proteins the cis state is populated to a small extent only. Adler and Scheraga (1990) showed by NMR that in heat-unfolded RNase A the nonnative trans isomers predominate at both Pro93 and Proll4. The Up molecules dominate in the unfolded state of proteins that have only tram-prolyl peptide bonds, such as lysozyme (Kato et ai, 1981, 1982), cytochrome c (Ridge el ai, 1981 Nall,... 

Probably not all proline residues are important for protein folding. Evidence for nonessential prolines came from a comparison of several homologous pancreatic RNases (Krebs et al., 1983, 1985) and cytochromes c (Babul et ai, 1978 Nall, 1990) that differ in the number of proline residues. Such prolines could be nonessential because they do not interfere with folding, or, alternatively, because they remain nativelike as regards isomeric state, after unfolding. 

Scheme 1. Kinetic model for the unfolding and isomerization of RNase Tl. This model is valid for unfolding only. The superscript and the subscript indicate the isomeric states of Pro39 and Pro55, respectively, in the correct, nativelike cis (c) and in the incorrect, nonnative trans (t) isomeric state. As an example, Ussc stands for an intermediate with Pro55 in the correct cis and Pro39 in the incorrect trans state. The two isomerizations are independent of each other, therefore the scheme is symmetric with identical rate constants in the horizontal and vertical directions, respectively. The given percentages for the individual unfolded species are estimates only. From Kiefhaber et al. (1990b,c).

Catahsis of Prolyl Isomerization in Unfolded Form of RCM-RNase Tl"... 

RS radicals enter the bilayer, then reach and isomerize the lipid double bonds from mto trans vide supra, Scheme 1). The conclusion that Met residues in RNAse A are the major source was further confirmed by monitoring of vesicle /-irradiation containing RNAse T1 (two S-S bridges, no Met residues) where the formation of the trans isomer was found only to 0.6% after 1 kGy. 

Fig. 9.3 The kinetic model for the slow prolyl isomerizations during refolding of RNase T1. U indicates the unfolded species, I the intermediates, and N is the native protein. Indices stand for the isomeric states of the prolines 39 and 55, respectively. Half-times given for the individual steps refer to folding conditions of 0.15 mol L-1 GdmCI, pH 5.0, 10°C [138,139].

In this section RNase A and RNase T1 are used as examples to illustrate the role of prolyl isomerizations for the unfolding and refolding of small single-domain proteins. Bovine pancreatic RNase A is selected because the history of the proline hypothesis and its experimental verification are closely related with this protein. The mechanism of RNase T1 folding is described because it is one of the major in vitro systems for investigating the function of prolyl isomerases as catalysts of proline-limited protein folding. 

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