DELETION OF THE OMEGA-LOOP IN THE ACTIVE-SITE OF STAPHYLOCOCCAL NUCLEASE .1. EFFECT ON CATALYSIS AND STABILITY

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 OMEGA-loop in the active site of E43 SNase having the sequence Glu43-Thr44-Lys45-His46-Pro47-Lys48-Lys49-Gly50-Val51-Glu52, where the gamma-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, we detected an unexpected deletion of residues 44-49 of the OMEGA-loop of E43 SNase in cassette mutagenesis experiments designed to randomize codons 44 and 45 in the OMEGA-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 OMEGA-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 OMEGA-loop generates a protein (E43 DELTA-SNase) having a partially solvent-exposed, surface beta-turn with the sequence Glu43-Gly50-Val51-Glu52; the structure of this beta-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 OMEGA-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 V(max). The deletion of the OMEGA-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 OMEGA-loop increases the stability of the folded form of E43 DELTA-SNase 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 OMEGA-loop are not 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 OMEGA-loop/beta-turn.