Ribonuclease reactions

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 experimental objective of the study was to obtain a series of stop-action photographs of ribonuclease A at work at atomic resolution. The strategy for such a program has been considered in detail by Fink and Petsko (1981), who treat such subjects as diffusional constraints and turnover rates, and in the preceding sections of this article. The ribonuclease reaction has a series of well-characterized, stable species which can be purchased, and crystals of the enzyme are large, well ordered, catalyt-ically active (Fink et al, 1984), and have as their natural mother liquor a cryoprotective solvent (Petsko, 1975). RNase thus represents the ideal system for a step-by-step analysis of an enzymatic catalytic pathway by the methods outlined above. 

Fig. 2. A pictorial representation of the ribonuclease reaction. The free enzyme (A) exists in two conformational states differing by small movements of the hinge region joining the two halves of the molecule. The substrate is bound (B) and a conformational change occurs closing the hinge (C). Concerted acid-base catalysis then occurs (D) products are formed ( ) the conformational change is reversed (F) and product(s) dissociate to give free enzyme.

A very different ribonuclease participates in the biosynthesis of all of the transfer RNAs of E. coli. Ribonuclease P cuts a 5 leader sequence from precursor RNAs to form the final 5 termini of the tRNAs. Sidney Altman and coworkers in 1980 showed that the enzyme consists of a 13.7-kDa protein together with a specific 377-nucleotide RNA component (designated Ml RNA) that is about five times more massive than the protein.779 Amazingly, the Ml RNA alone is able to catalyze the ribonuclease reaction with the proper substrate specificity.780 7823 The protein apparently accelerates the reaction only about twofold for some substrates but much more for certain natural substrates. The catalytic center is in the RNA, which functions well only in a high salt concentration. A major role of the small protein subunit may be to provide counterions to screen the negative charges on the RNA and permit rapid binding of substrate and release of products.783 Eukaryotes, as well as other prokaryotes, have enzymes similar to the E. coli RNase R However, the eukaryotic enzymes require the protein part as well as the RNA for activity.784... 

Fig. 2.15. Hydroylysis of cytidine-2, 3 -phosphate by ribonuclease (reaction mechanism according to Findlay, 1962)...

Memfield successfully automated all the steps m solid phase peptide synthesis and computer controlled equipment is now commercially available to perform this synthesis Using an early version of his peptide synthesizer m collaboration with coworker Bemd Gutte Memfield reported the synthesis of the enzyme ribonuclease m 1969 It took them only SIX weeks to perform the 369 reactions and 11 391 steps necessary to assemble the sequence of 124 ammo acids of ribonuclease... 

Further computational studies on adenines and adenosines concern the reaction mechanism of ribonuclease A with cytidyl-3,5 -adenosine [99BP697] and the molecular recognition of modified adenine nucleotides [99JMC5338]. 

Finally, students can be critics of published work, and perhaps have already encountered papers in the literature with questionable features. I invite reference to the paper, On the Mechanism of Catalysis by Ribonuclease Cleavage and Isomerization of the Dinucleotide UpU Catalyzed by Imidazole Buffers [Anslyn, E. Breslow, R. J. Am. Chem. Soc. 1989, III, 4473 1482]. A useful exercise is to list any flaws. Any such criticisms can then be compared with those raised in the article, Imidazole Buffer-Catalyzed Cleavage and Isomerization Reactions of Dinucleotides The Proposed Mechanism Is Incompatible with the Kinetic Measurements [Haim, A. J. Am. Chem. Soc. 1992,114, 8383-8388]. 

Isab, A.A. and Sadler, P.J. (1977) Reactions of gold(III) ions with ribonuclease A and methionine derivatives in aqueous solution. Biochimica et Biophysica Acta, 492, 322-330. 

Adams, G.E., Bisby, RH., Cundall, R.B., Redpath, J.L. and Willson, RL (1972). Selective free radical reactions with proteins and enzymes. The inactivation of ribonuclease. Radiat. Res. 49, 290-298. 

Lichtin, N.W. et al. (1972). Fast consecutive radical processes within the ribonuclease molecule in aqueous solution. II. Reaction with OH and e(lq ). Biochim. Biophys. Acta 276, 124-131. 

In many enzymes, the value of kc-JK lies between 108 and 109 M s. The value for L 19 RNA is 103 M 1 s1, i.e., five orders of magnitude lower than for protein enzymes with high catalytic activity. However, L 19 RNA does compare in its efficiency to the enzyme ribonuclease A. The capabilities of ribozymes referred to above dealt solely with interactions of RNA (i.e., ribozymes) with RNA molecules. In a (hypothetical) RNA world, they would, however, need to be capable of doing much more, e.g., carrying out reactions at the carbon skeletons of biomolecules. 

Cytokine profiling has also been measured as a function of changes in cytokine mRNA expression using either reverse transcription polymerase chain reaction (RT-PCR) [87, 91-93] or ribonuclease protection assay (RPA) [94-97], Measurement of cytokine transcripts by RT-PCR revealed that prolonged exposure to TMA induced increased levels of IL-4 mRNA expression compared with treatment with DNCB [87,92-93]. However, expression of the type 1 cytokine IFN-y by DNCB-activated LNC was variable and failed to discriminate between contact and respiratory allergens [87,91,93). A similar profile was observed for freshly isolated tissue analyzed by RPA. This somewhat less... 

J.-R. Garel and R. L. Baldwin, Both the fast and slow refolding reactions of ribonuclease A yield native enzyme, Proc. Natl. Acad. Sci. U.S.A. 70, 3347-3351 (1973). 

Since the rate constants of bimolecular diffusion-limited reactions in isotropic solution are proportional to T/ these data testify to the fact that the kt values are linearly dependent on the diffusion coefficient D in water, irrespective of whether the fluorophores are present on the surface of the macromolecule (human serum albumin, cobra neurotoxins, proteins A and B of the neurotoxic complex of venom) or are localized within the protein matrix (ribonuclease C2, azurin, L-asparaginase).1 36 1 The linear dependence of the functions l/Q and l/xF on x/t] indicates that the mobility of protein structures is correlated with the motions of solvent molecules, and this correlation results in similar mechanisms of quenching for both surface and interior sites of the macromolecule. 

An example of this effect is provided by ribonuclease A (RNase A). At pH 8 and 37°, the rate of deamidation of Asn67 was more than 30-fold lower in the native than in the unfolded protein [111]. Deamidation of the native RNase A was also ca. 30-fold slower than of an octapeptide whose sequence is similar to that of the deamidation site, although the reaction mechanisms were similar [108][123],... 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

List of Abbreviations cDNA, complementary DNA ddH20, double-distilled H2O dNTP, deoxyribonu-cleotide triphosphate EDTA, ethylenediaminetetraacetic acid MgCl2, magnesium chloride mRNA, messenger ribonucleic acid NaOH, sodium hydroxide PCR, polymerase chain reaction qRT PCR, quantitative reverse transcriptase polymerase chain reaction RNase, ribonuclease RT PCR, reverse transcriptase polymerase chain reaction UTR, untranslated region... 

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