Kinetic studies ribonuclease

They have studied the variation of this coefficient with urea concentration, temperature, and pH, and have also studied the ultraviolet absorption and kinetics of the reversal of denaturation which occurs when a solution of ribonuclease in concentrated urea is diluted with several volumes of aqueous buffer. Similar kinetic studies on other proteins should afford data of great value. 

A. Tekos, A. Tsagla, C. Stathopoulos, and D. Drainas, Inhibition of eukaryotic ribonuclease P activity by aminoglycosides kinetic studies, FEBS Lett., 485 (2000) 71-75. 

Fig. 9-7 A schematic mechanism for the hydrolysis of a dinucleoside by ribonuclease A. The various conformational states inferred to be present from kinetic studies are shown. In this mechanism PypN is a pyrimidine 3 5 -nucleoside, Py 2 3 p is a pyrimidine 2 3 cyclic phosphate and E is the enzyme. The primes designate different conformational states of the enzyme and enzyme-substrate complexes.

Since there appear to be internal interactions in ribonuclease it was important to determine whether these involved hydrogen bonds and, if so, how many hydrogen bonds. For this purpose kinetic studies of deuterium-... 

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. 

The preceding summary and Fig. 20 present a frame-by-frame account of the pathway for ribonuclease catalysis, based predominandy on knowledge of the structures of the various intermediates and transition states involved. The ability to carry out such a study is dependent on three critical features (1) crystals of the enzyme which diffract sufficiently well to permit structural resolution to at least 2 A (2) compatibility of the enzyme, its crystals, and its catalytic kinetic parameters with cryoenzymology so as to permit the accumulation and stabilization of enzyme-substrate complexes and intermediates at subzero temperatures in fluid cryosolvents with crystalline enzyme and (3) the availability of suitable transition state analogs to mimic the actual transition states which are, of course, inaccessible due to their very short lifetimes. The results from this investigation demonstrate that this approach is feasible and can provide unparalleled information about an enzyme at work. 

The ultimate objective of an X-ray cryoenzymological study is the mapping of the structures of all kinetically significant species along the reaction pathway. In the case of ribonuclease A this has been largely achieved, as described above. Other enzymatic reactions now await application of the same techniques. Unfortunately, not all crystalline enzymes lend themselves to study by this method. In some cases it may be impossible to find a suitable cryoprotective mother liquor in others, the reaction may occur too rapidly at ordinary temperature. A reaction with Acat of 10 seconds and an activation enthalpy of —6 kcal mol will not be quenched even at — 75°C. The approach we have described in this article can be applied to only a small number of enzymes. Two likely candidates for successors to ribonuclease are the enzymes yeast triosephosphate isomerase and porcine pancreatic elastase. 

These points arc illustrated by the elegant studies of Nelson and Hummel (1962) on the kinetics of denaturation of ribonuclease by urea. As is shown in Fig. 16, the difference spectrum generated with ribonuclease using 7 M urea versus aqueous reference solvent is markedly time-dependent. Difference spectra taken shortly after mixing protein and urea solvent reflect the refractive index red-shift of urea on the tyrosyl and phenylalanyl groups of the protein in its native conformation. As the urea denaturation... 

The one-gene-one-enzyme concept did imply that the primary structure of the peptide determines the secondary, tertiary, and quaternary structure, and this was established by Anhnsen (1973) by an analysis of the mutant ribonuclease and by the study of chemical modification as well as the denaturation and renaturation kinetics of this enzyme (Anhnsen 1973). 

Panick, G., Winter, R. Pressure-induced unfolding/refolding of ribonuclease A static and kinetic Fourier transform infrared spectroscopy study, Biochem. 39 (2000) 1862-1869. 

As the result of our kinetic evidence, we had to propose a mechanism (Scheme 2) for the process of RNA cleavage by imidazole buffer, and other buffers such as morpholine, in which the first function of the BIP" is to protonate the phosphate group. We have proposed [2] that the enzyme ribonuclease A uses a related mechanism (Scheme 3)— but simultaneous instead of sequential—and cited some physical and calculational evidence in support of this proposal. We have used the evidence from this mechanistic work to redesign a catalyst that imitates some aspects of the enzyme mechanism. However, we were also forced to think about the properties of the isomeric 2, 5 -linked nucleic acids whose formation was a key piece of evidence in the mechanistic studies. We will first describe work fliat addresses this question. 

One of the most effective uses of optical rotation is in the study of kinetics and equilibria of protein denaturation. For example, Harrington and Schellman (1956) found that the values of [ ]d of ribonuclease become more negative if the protein is heated at pH 6.5 (see Fig. 101). This curve is... 

Slim, G. and Gait, M J (1991) Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes Nucl. Acids Res 19,1183-1188 Pieken, W A, Olsen, D. B, Benseler, F., Aurup, H, and Eckstein, F (1991) Kinetic characterization of ribonuclease-resistant 2 -modified hammerhead ribozymes. Science 252, 314-317... 

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