The high-resolution X-ray structure of wild-type staphylococcal nuclease (E43 SNase) suggests that Glu 43 acts a general basic catalyst to assist the attack of water on a phosphodiester substrate [Loll, P., & Lattman, E. E. (1989) Proteins Struct., Funct., Genet. 5, 183]. Glu 43 is located at the base of the solvent-exposed and conformationally mobile Ω-loop in the active site of E43 SNase having the sequence Glu43-Thr44-Lys45-His46-Pro47-Lys48-Lys49-Gly5o-Val5-Glu52, where the γ-carboxylate of Glu 52 is hydrogen bonded to the amide hydrogen of Glu 43. With a metabolic selection for SNase activity produced in an Escherichia coli host host, we detected an unexpected deletion of residues 44–49 of the Ω-loop of E43 SNase in cassette mutagenesis experiments designed to randomize codons 44 and 45 in the Ω-loop and increase the activity of the previously described E43D mutation (D43 SNase). A high-resolution X-ray structure of D43 SNase has revealed that the E43D substitution significantly changes the structure of the Ω-loop, reduces the interaction of the essential Ca2+ ion with its active-site ligands, and diminishes the network of hydrogen-bonded water molecules in the active site [Loll, P., & Lattman, E. E. (1990) Biochemistry 29, 6866]. This deletion of six amino acids from the Ω-loop generates a protein (E43 ΔSNase) having a partially solvent-exposed, surface β-turn with the sequence Glu43-Gly50-Val51;-Glu52; the structure of this β-turn is addressed in the following article [Baldisseri et al. (1991) Biochemistry (following paper in this issue)]. The deletion of six amino acids from the Ω-loop starting with residue 43 is less damaging to catalysis than the excision of a single methylene group from residue 43 (in D43 SNase) as assessed by measurements of Vmax. The deletion of the Ω-loop and the E43D substitution are nonadditive mutations, presumably the result of the amount of rate acceleration possible with general basic catalysis and a conformational alteration produced by the E43D substitution. The deletion of the Ω-loop increases the stability of the folded form of E43 ΔSNase relative to the E43 SNase by 2.5 kcal/mol. In contrast to the E43D substitution [Hibler, D. W., Stolowich, N. J., Reynolds, M. A., Gerlt, J. A., Wilde, J. A., & Bolton, P. H. (1987) Biochemistry 26, 6278], the changes in catalysis and stability induced by deletion of the Ω-loop are nof accompanied by significant changes in either the chemical shifts or the interresidue nuclear Overhauser effects of aromatic and upfield-shifted aliphatic residues both in the hydrophobic core and near the base of the Ω-loop/β-turn.
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