Ribonuclease catalytic activity

S. Moore and W. H. Stein (Rockefeller, New York) contributions to the understanding of the connection between chemical structure and catalytic activity of the active centre of the ribonuclease molecule. 

For many solubilized enzymes the greatest catalytic activity and/or changes in conformation are found at R < 12, namely, when the competition for the water in the system between surfactant head groups and biopolymers is strong. This emphasizes the importance of the hydration water surrounding the biopolymer on its reactivity and conformation [13], It has been reported that enzymes incorporated in the aqueous polar core of the reversed micelles are protected against denaturation and that the distribution of some proteins, such as chymotrypsine, ribonuclease, and cytochrome c, is well described by a Poisson distribution. The protein state and reactivity were found markedly different from those observed in bulk aqueous solution [178,179],... 

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. 

Klink TA, Woycechowsky KJ, Taylor KM, et al. Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A. Eur. J. Biochem. 2000 267 566-572. 

Divalent metal ions are essential for ribonuclease H activity. Two Mn(II) ions have been located in the catalytic site of ribonuclease H domain of HIV-1 reverse transcriptase in close proximity to the four acidic residues Asp443, Glu478, Asp498, and Asp549 after soaking crystals in 45 mM MnCl2 (406). 

The S-ribonuclease is the complex formed between an eicosapeptide and the S-RNAse. While replacement of various amino acids by fluorinated analogues does not modify the activity of the native complex, replacement of His-12 by 4-F-His has a strong influence. Indeed, the S-ribonuclease, formed between the bovine pancreatic S-RNAse and the fluoro peptide that contains 4-F-His, has no more catalytic activity, but it is stable. This loss of enzymatic activity is probably due to the significant lowering of the pAia of the catalytic His (2.5 units), which results from the presence of the fluorine atom. It is known that histidine plays an important role in nucleophilic and acid-base processes, which are connected to the catalytic activity of numerous enzymes. 

Stanford Moore United States chemical structme and catalytic activity of ribonuclease... 

Ribonuclease A was the first enzyme to be synthesized in the laboratory. Fully active ribonuclease has been synthesized,752 as have new modified enzymes. For example a 63-residue peptide made up of five segments of the native RNase sequence retained measurable catalytic activity.753 Using total synthesis, unnatural amino acids, such as 4-fluorohistidine, have been incorporated at specific positions in RNAse.752... 

The enzyme consists of a single polypeptide chain of Mr 13 680 and 124 amino acid residues.187,188 The bond between Ala-20 and Ser-21 may be cleaved by subtilisin. Interestingly, the peptide remains attached to the rest of the protein by noncovalent bonds. The modified protein, called ribonuclease S, and the native protein, now termed ribonuclease A, have identical catalytic activities. Because of its small size, its availability, and its ruggedness, ribonuclease is very amenable to physical and chemical study. It was the first enzyme to be sequenced.187 The crystal structures of both forms of the enzyme were solved at 2.0-A resolution several years ago.189,190 Subsequently, crystal structures of many complexes of the enzyme with substrate and transition analogues and products have been solved at very high resolution.191 Further, because the catalytic activity depends on the ionizations of two histidine residues, the enzyme has been extensively studied by NMR (the imidazole rings of histidines are easily studied by this method—see Chapter 5). 

Ribonuclease-S can be separated into S-peptide [residues 1-20 (21)] and S-protein [residues 21 (22)-124] by precipitation with trichloroacetic acid 73) or better, Sephadex chromatography in 5% formic acid 83). The best preparations of these components show no detectable hydrolytic enzymic activity and little if any transphosphorylation activity (see Section VI). Isolated S-peptide appears to have no regular secondary structure 83, 84) or 10-20% helicity 85, 86). (These slightly different interpretations are based on almost identical CD data.) When equimolar amounts of S-protein and S-peptide are mixed at neutral pH and room temperature or below, essentially full catalytic activity is recovered 73, 87). A schematic diagram is shown in Fig. 7. For a detailed summary of the preparative procedures see Doscher 88). 

The Anfinsen experiment raises the question Must we always use x-ray diffraction to determine a protein s structure In the case of ribonuclease the structure is known from x-ray diffraction, but at the time of Anfinsen s investigation it was not. He relied heavily on the fact that the reconstituted enzyme had the same activity as the native enzyme. This remains an acceptable approach as long as one is working with an enzyme that has a demonstrable catalytic activity, but what of the many proteins that do not ... 

Leon-Lai, C.H., M.J. Gresser, and A.S. Tracey. 1996. Influence of vanadium(V) complexes on the catalytic activity of ribonuclease A. The role of vanadate complexes as transition state analogues to reactions at phosphate. Can. J. Chem. 74 38 -8. 

Our understanding of the principles underlying the catalytic activity of enzymes has increased greatly in recent years. Enzymes are proteins and we know now that the polypeptide chain of a globular protein molecule will assume spontaneously a well-defined conformation (3). This native conformation of the macromolecule is essential to the function of the enzyme. Recent studies show that enzyme crystals, into which substrate molecules may penetrate by diffusion, have similar catalytic characteristics as enzyme solutions (4) and this result tends to strengthen our belief that the conformation of the enzyme molecule, as deduced from X-ray diffraction studies of enzyme crystals, is identical, or at least very similar, to the conformation responsible for the catalytic activity under physiological conditions. The structures of some enzymes, e.g., lysozyme (5), ribonuclease (6), and carboxypeptidase A (7), have been determined they are all consistent with the general belief that... 

Ribonuclease P (RNase P) consists of both protein and an RNA component that has catalytic activity. RNase P functions in eukaryotic cells to process the 5 end of precursor tRNA molecules. RNase P also can be directed to cleave any RNA molecule when the target is complexed with a short complementary oligonucleotide called an external guide. 

The 5 end is created by ribonuclease P, which cleaves to leave a phosphate on the 5 terminal G. This enzyme creates the 5 terminus of all tRNA molecules. It is not clear what structural features are recognized by RNase P, for different sequences are contained in the cleavage sites. Ribonuclease P consists of one RNA molecule of 377 nucleotides and one protein molecule with Mr of about 20,000. Both components are necessary for full catalytic activity, but under nonphysiological conditions the RNA molecule alone can... 

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