RNase kinetics

The association between RNase A and 3 -UMP or 3 -dUMP has been studied by P n.m.r. and kinetic methods, respectively. In both cases the participation of two dissociable groups at the active site of the enzyme was demonstrated, in agreement with n.m.r. and A -ray - studies on the binding of 3 -CMP to RNase. In the binding of Tj RNase to purine nucleotide monophosphates, the phosphate group appears to have an important effect while the ribose ring is relatively unimportant. ... 

Despite considerable biochemical work, high-resolution crystal structure determination of native RNase A and S, and some medium-resolution studies of RNase A-inhibitor complexes, a number of questions existed concerning the details of the catalytic mechanism and the role of specific amino acids. Study of the low-temperature kinetics and three-dimensional structures of the significant steps of the ribonuclease reaction was designed to address the following questions. 

RNase M Np, Non-s Zn2+, Cu2+ other effectors kinetic data photooxidation inhibition by nucleotides 166)... 

In a detailed study of the equilibria involved in the urea transition, Tanford (354) showed that a two-state process could not explain the RNase data and that the cooperative units, whose unfolding was reflected in the measurements, must be much less than the total molecule. Each unit was probably not more than one-third of the total molecule. This conclusion was based on Tanford s theory and the data of Nelson and Hummel (351) and of Foss and Schellman (355). The midpoint of the transition at room temperature in 0.1 M KC1 and neutral pH is about 6 M urea. Barnard (356) found a midpoint at 6.7 M urea at pH 7 and 25°C. By fluorescence the midpoint was about 6.5 M urea (308). Between 12 and 16 molecules of urea per molecule of protein appeared to be involved in the transition (356), 12 being the kinetic order of the unfolding reaction and 16 being the value derived from the slope of the equilibrium curve. Again evidence for multiple states was presented. 

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). 

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).

Fig. 7. Oxidative refolding of reduced RNase Tl. Reoxidation conditions were 0.1 M Tris-HCl, pH 7.8, 0.2 Af guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 nM RNase Tl at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm ( ), by an unfolding assay (Kiefhaber el ai, 1990b) that measures the formation of native protein molecules (A), and by the increase in the intensity of the band for native RNase Tl in native polyacrylamide gel electrophoresis ( ). Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (O). The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

Fig. 8. Acceleration of the oxidative refolding of RNase T1 by PPI and PDI. The increase in fluorescence at 320 nm is shown as a function of the time of reoxidation. The final conditions were 2.5 fiM RNase T1 in 0.1 Af Tris-HCl, 0.2 M GdmCl, 2 mM EDTA, 3 mAf glycine, 0.4 mAf oxidized glutathione, and 4 mAf reduced glutathione at pH 7.8 and 25°C. Reoxidation ( ) in the absence of PPI and PDI, (O) in the presence of 1.4 tiM PPI, (A) in the presence of 1.6 fiM PDI, and (A) in the presence of both 1.6 fiM PDI and 1.4 /uAf PPI. In all experiments more than 90% of the observed kinetics were well approximated by single first-order processes, as indicated by the continuous lines. The respective time constants (t) are ( ) t = 4300 sec, (O) r = 2270 sec, (A) t = 1500 sec, (A) T = 650 sec. In all cases the initial fluorescence signal was about 10% of the final emission of the native protein. From Schonbrunner and Schmid (1992).

The specific activity and thermodynamic stability of the (C2A, ClOA) mutant confirm that the Cys-2 to Cys-10 disulfide bond imparts thermodynamic stability but has litde effect on catalytic activity. Hence this mutant was selected as the starting point for constructing a circularly permuted form of RNase-Tl so that as short a linker as possible could be used to bridge the original N- and C-termini. The activity and stability of the circularly permuted variant indicate that it adopts an overall tertiary fold very similar to that of the native protein. Therefore, transposing the first 34 residues to the C-terminus has little effect on the overall folding to the final tertiary structure. The real effect, however, may be more evident in the kinetics of the specific folding pathway. 

Figure 14.10. Elution profile of RNase as a function of free [5 -TMP], using an affinity column with immobilized 5 -TMP. The concentrations of 5 -TMP in the mobile phase were 5.0 x 10 4Af, 4.0 x 10 4Af, 3.0 x 10 4Af,2.0 x 10 4M, 1.0 x 10 4M, and 7.5 x 10 5M for the earliest to the latest eluted fractions, respectively. The inset shows the plot according to Eq. 14.24 that was used to determine the association constant for the RNase-5 -TMP binding reaction.11 [Reprinted, with permission, from B. M. Dunn and I. M. Chaiken, Biochemistry 14 (No. 11), 1975, 2343-2349. Evaluation of Quantitative Affinity Chromatography by Comparison with Kinetic and Equilibrium Dialysis Methods for the Analysis of Nucleotide Binding to Staphylococcal Nuclease . 1975 by American Chemical Society.]...

RNA interaction and therefore cannot be recommended for probing experiments (e.g. RNase SI and RNase U2 have a pH optimum of 4.5 which prevents or destabilises protein-RNA complex formation). RNase A has a high affinity for a pyrimidine-adenosine stretch (particularly UA), so it can therefore be difficult to obtain single-hit kinetics (except for the UA sequence). Furthermore, RNase A exhibits an endogenous helix unfolding property which makes structure assignment difficult. RNases such as RNase CL3 and a-sarcin are inhibited by Mg2+ which is required for the stability of many complexes and also for proper folding of RNA. 

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].

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