SNase active site

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

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. 

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 ... 

Staphylococcal nuclease (SNase) is one of the most powerful enzymes known in terms of its rate acceleration, with a catalytic rate that exceeds that of the non-enzymatic reaction by as much as 1016.211 This enzyme is a phosphodiesterase, and utilizes a Ca2+ ion for catalysis to hydrolyze the linkages in DNA and RNA. In addition to the metal ion, the active site has two Arg residues in a position to interact with the phosphoryl group, and a glutamate. X-ray structures212 215 of SNase have been solved for the wild-type enzyme and mutants, but the exact roles of active-site residues are still uncertain. SNase cleaves the 5 O-P nucleotide bond to yield a free 5 -hydroxyl group (Fig. 30). 

The rate-limiting step for the hydrolysis of single-stranded DNA by SNase was subsequently shown to be product dissociation, and substrate binding is diffusion-controlled when the pH is >7.3.219 The pH optimum is between 8.6 and 10.3, and the enzyme is routinely assayed at pH 9.5. Thus, under these conditions, kinetic studies cannot reveal the effect of mutations on the chemical step. On the basis of the dependence of rate on pH it was proposed that Glu-43 does not act as a general base.219 It was also proposed that residues in a protein loop near the active site may be involved in the physical process that is the rate-determining step.219... 

The putative general base catalyst, Glu-43, and electrophilic catalysts, Arg-35 and Arg-87, have been specifically mutated to Asp (9/, 92) and Lys (93) residues, respectively, in the author s laboratory to assess the roles of these amino acid residues in catalysis. Other amino acids were also introduced at these positions, but the present discussion will briefly outline the results obtained with the conservative substitutions. As mentioned previously, the rate acceleration characteristic of the SNase-catalyzed hydrolysis of DNA is approximately 10. The Asp substitution for Glu-43 (E43D) decreased the catalytic efficiency approximately 10, and the Lys substitutions for Arg-35 (R35K) and Arg-87 (R87K) decreased the catalytic efficiency approximately 10 and 10, respectively. While such decreases in catalytic efficiency have been used to describe quantitatively the roles of various active site residues in catalysis, such interpretation is clearly unwarranted in the case of these active site mutants of SNase. The melting temperatures of all three of these mutant enzymes differ significantly... 

Figure 5.1 Stereo view of the active site of SNase. Coordinates are from the dataset 2SNS [ 15] of the Brookhaven Protein Databank. The positions of the two Ca ligated waters and the reacting water molecule have been indicated with small circles.

The same calculation is then performed with the ion in the active site of SNase for each of the three states 2-.3- Hence, with the previously... 

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