SNase

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 Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity.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. 

Staphylococcal nuclease (SNase) is a single-peptide chain enzyme consisting of 149 amino acid residues. It catalyzes the hydrolysis of both DNA and RNA at the 5 position of the phosphodiester bond, yielding a free 5 -hydroxyl group and a 3 -phosphate monoester... 

FIGURE 8.1. The structure of the active site of SNase with a bound inhibitor that is used as a model for the substrate. 

The overall catalytic rate constant of SNase is (see, for example, Ref. 3) kcat — 95s 1 at T = 297K, corresponding to a total free energy barrier of Ag at = 14.9 kcal/mol. This should be compared to the pseudo-first-order rate constant for nonenzymatic hydrolysis of a phosphodiester bond (with a water molecule as the attacking nucleophile) which is 2 x 10 14 s corresponding to Ag = 36 kcal/mol. The rate increase accomplished by the enzyme is thus 101S-1016, which is quite impressive. 

FIGURE 8.2. The resonance structures for the proposed mechanism of SNase. 

The determination of the AG - s depends, of course, on the choice of the reference reaction in solution. For instance, when one states that the rate enhancement by SNase is 1016, one makes the implicit assumption of the reference reaction being... 

FIGURE 8.3. The energetics of an hypothetical reference reaction that corresponds to the assumed mechanism of SNase but occurs in a solvent cage. 

FIGURE 8.5. Three snapshots from the trajectories that lead from the ground state to the transition state in the catalytic reaction of SNase. 

Calculations of the actual dependence of the activation barrier, Ag, on the metal size in the active site of SNase are summarized in Fig. 8.10. The results reflect mainly the energetics of i//2 and ij/3, since the dependence on the ionic radius in is found to be rather small. 

The finding that SNase appears to have its turnover optimum for the ion which it uses in nature may, of course, not be considered terribly surprising. Flowever, the free-energy relationships leading to a rate optimization are quite interesting and point toward more general features pertaining also to... 

Barium, effectiveness as cofactor for, see also Enzyme cofactors phospholipase, 204 SNase, 200-204 Bond-breaking processes, 12 potential surfaces for, 13-14, 18-20 in solutions, 22,46-54... 

Catalysis, general base (Continued) in phosphodiester hydrolysis by SNase, 190... 

Enzyme active sites, 136,148, 225. See also Protein active sites in carbonic anhydrase, 197-199 in chymotrypsin, 173 in lysozyme, 153, 157 nonpolar (hypothetical site), 211-214 SNase, 189-190,190 steric forces in, 155-158, 209-211, 225 in subtilisin, 173 viewed as super solvents, 227 Enzyme cofactors calcium ... 

Metal ions, effect of size, 200-205 Metalloenzymes, see also Enzyme cofactors classification of, by cofactor and coupled general base, 205-207, 206 electrostatic interactions in, 205-207 SNase, 189-197... 

SNase, see Staphylococcal nuclease (SNase) S,v2 reactions, see Substitution reactions, nucleophilic (Sw2)... 

Because the molar volume of an unfolded protein is less than that of the native state, increasing pressure leads to denaturation (Gross and Jaenicke, 1994). Royer and co-workers have employed high-pressure SAXS to monitor the pressure-induced unfolding of Snase. They find that the unfolded ensemble achieves a pressure-independent Rg of... 

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