Staphylococcal nuclease mechanism

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

Tucker, P.W., Hazen, E.E., Colton, F.A. Staphylococcal nuclease reviewed a prototypic study in contemporary enzymology. III. Correlation of fhe three-dimensional structure with the mechanisms of enzymatic action. Mol. Cell. Biochem. 

It should be noted that the unfolding kinetics can sometimes involve quite complex unfolding schemes of different substates in equilibrium with the native state. Staphylococcal nuclease is an example of such behavior, known to unfold with three different substates that exhibit an equilibrium that does not appear to shift with temperature.49 Irreversible aggregation processes of proteins have been known to involve first- or second-order reactions.132141 The mechanism of recombinant human interferon-y aggregation is an example where thermodynamic and kinetic aspects of the reaction provided a powerful tool for understanding the pathway of instability and permitted a rationale for screening excipients that inhibited the process.141... 

Cotton, F. A., Hazen, E. E., Jr., and Legg, M. J. (1979). Staphylococcal nuclease Proposed mechanism of action based on structure of enzyme-thymidine 3, 5 -bisphosphate-calcium ion complex at 1.5-A resolution. Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555. 

Mildvan, A. S., and Serpersu, E. H. (1989). Genetic alteration of active site residues of staphylococcal nuclease Insights into the enzyme mechanism. In Metal Ions in Biological Systems (H. Sigel and A. Sigel, eds.), pp. 309-334. Dekker, New York. 

Figure 12-29 Drawing showing the hydrogen-bonding interactions between the guanidinium ions of arginines 35 and 87 of the micrococcal (staphylococcal) nuclease with the 5 -phosphate of the inhibitor thymidine 3, 5 -diphosphate in the complex of E + I + Ca2+. A possible mechanism is illustrated. A hydroxyl ion bound to Ca2+ carries out an in-line attack on the phosphorus. See Libson et al.S26...

The apparent usefulness of the modeling approach suggested that possible active site interactions important in understanding the mode of action of the well-characterized enzymes, ribonuclease (16) and staphylococcal nuclease (17). may be revealed. Both have been the subject of extensive crystallographic studies (18,19) with suitable inactive substrates in place. We considered the first step of hydrolytic action of ribonuclease (RNase) on the dinucleotide substrate uridylyl-(3 -5 )-adenosine(UpA). Our results (20) on the enzyme mechanism were consistent with the main features summarized by Roberts et al (21). The first step is a transphosphorylation leading to cleavage "oT the phosphodiester... 

Abstract. Walter Kauzmann stated in a review of protein thermodynamics that volume and enthalpy changes are equally fundamental properties of the unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behaviour (Nature 325 763-764, 1987). While the thermodynamic basis for pressure effects has been known for some time, the molecular mechanisms have remained rather mysterious. We, and others in the rather small field of pressure effects on protein structure and stability, have attempted since that time to clarify the molecular and physical basis for the changes in volume that accompany protein conformational transitions, and hence to explain pressure effects on proteins. The combination of many years of work on a model system, staphylococcal nuclease and its large numbers of site-specific mutants, and the rather new pressure perturbation calorimetry approach has provided for the first time a fundamental qualitative understanding of AV of unfolding, the quantitative basis of which remains the goal of current work. 

RA. Cotton, E.E. Elazen, M.J. Legg, Staphylococcal Nuclease - Proposed Mechanism of Action Based on Structure of Enzyme-Thymidine 3, 5 -Bisphophate-Calcium ion Complex at 1.5 A Resolution , Proc. Natl. Acad. Sci. USA, 76, 2551 (1979)... 

Judice JK, Gamble TR, Murphy EC, de Vos AM, Schultz PG. Probing the mechanism of staphylococcal nuclease with unnatural amino acids kinetic and structural studies. Science 1993 261 1578-1581. 

Figure 15. Reaction mechanism for staphylococcal nuclease, (a) gcncral-basc catalysis, (b) nucleophilic attacl by a hydroxyl anion, (c) cleavage of the 5 0-P bond.

Weiss and coworkers pioneered in using single-molecule FRET to study the conformational dynamics and reaction mechanism of staphylococcal nuclease.85 Lu and coworkers used it to study the conformational dynamics of T4 lysozyme during catalysis.58 Hammes, Benkovic, and coworkers studied dihydrofolate reductase,25 the enzymes involved in T4 primosome86 and replisome.87 88 Yang and coworkers studied adenylate kinase.89 Here, we use the T4 lysozyme study to illustrate the approach.5... 

Since metal coordination or immobilization of the transferred phosphoryl group by multiple hydrogen bonds would inhibit the formation of a metaphosphate intermediate in an S l mechanism and would facilitate nucleophilic attack in an Sy2 mechanism, the latter process seems likely for the reactions catalyzed by staphylococcal nuclease, DNA polymerase, pyruvate kinase, fructose diphosphatase, phosphoglucomutase, (Na + K) ATPase and possibly PEP carboxylase. In creatine kinase where an S l mechanism is possible, the enzyme would have to prevent access of nucleophiles other than ADP and creatine to the reactive metaphosphate intermediate. 

Genetic Alteration of Active Site Residues of Staphylococcal Nuclease Insights into the Enzyme Mechanism... 

Earlier suggestions of stereoelectronic control of acetal cleavage for the lysozyme reaction Gorenstein, D. G., Findlay, J. B., Luxon, B. A., Kar, D. (1977). Stereoelectronic control in carbon-oxygen and phosphorus-oxygen bond breaking processes. Ah initio calculations and speculations on the mechanism of action of ribonuclease A, staphylococcal nuclease, and lysozyme. Journal of the American Chemical Society, 99, 3477. 

The masters of catalysis are enzymes. Enzymes are biomolecules typically based on proteins and often associated with small organic molecules or metal ions known as cofactors. In recent years it has become clear that RNA molecules can also catalyze important reactions, and such catalytic RNA molecules are referred to as ribozymes. Our focus here, however, will be on the more well known, protein-based enzymes, which mediate the overwhelming majority of biochemical transformations. These are nature s catalysts, and they can be incredibly efficient. As just one example, the hydrolysis of a phosphoester such as that used to link nucleotides together in DNA is estimated to have a half-life of hundreds of millions of years in water at neutral pH. Yet, the enzyme staphylococcal nuclease can catalyze this hydrolysis reaction with a half-life of a few minutes. Since this is a physical organic textbook, not a biochemistry textbook, we do not look at the structures of enzymes and how they are formed. Instead, we simply focus upon the mechanisms and kinetics of enzymatic catalysis. 

Staphylococcal nuclease has been extensively used as a model protein for the study of enzyme mechanisms, stability, and the kinetics of (re)folding. In recombinant DNA technology, SNase is primarily used as a nonspecific endonuclease, as is DNase I which generates 5 -phosphonucleotides (Section I, this chapter). Staphylococcal nuclease has also served as the prototype model for a new generation of nucleases called hybrid nucleases, which can cleave DNA and RNA with tailored site specificities. 

Most of the main concepts regarding the mechanisms of protein folding accepted today originated from both theoretical conformational computations and the determination of structures at atomic resolution. The amount of experimental data is still insufficient to allow a high degree of generalization. The number of known proteins for which a detailed and complete study of the folding process is available, remains indeed very small. Well documented systems such as ribonuclease, staphylococcal nuclease, BPTI, lysozyme, serine proteases, and few other proteins are used frequently as examples in the discussion that follows. 

The mechanism by which staphylococcal nuclease catalyzes the hydrolysis of single-stranded RNA and DNA is uncertain, despite the fact that the complete amino acid sequence (Cone et al., 1971) and a 1.5-A X-ray structure (Cotton et al, 1979) are available. In order to provide addition ... 

The nuclease requires Ca + ions for activity, with no other divalent metal ion being able to support catalysis. A large number of other phospodiester-ases have been found to be dependent on divalent metal ions for activity, including the restriction endonuclease icoRI (Barton et al, 1982). Thus elucidation of the mechanism of the reaction catalyzed by staphylococcal nuclease may provide important clues to the mechanisms of the other metal-dependent phosphodiesterases. Fortunately, staphylococcal nuclease will catalyze, albeit at a low rate, the hydrolysis of a number of mononucleotide esters (Cuatrecasas et al., 1969), including thymidine 5 -(4-nitro-phenyl phosphate) this ester is hydrolyzed to thymidine and 4-nitrophenyl phosphate. We have determined the stereochemical course of the hydrolysis of thymidine 5 -(4-nitrophenyl [ 0, 0]phosphate) and interpreted the result in terms of the structure of the active site of the enzyme. 

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