Deletion of the Ω-Loop in the Active Site of Staphylococcal Nuclease. 1. Effect on Catalysis and Stability

Leslie B. Poole, A. Deborah Loveys, Stephen P. Hale, John A. Gerlt, Susan M. Stanczyk, Philip H. Bolton

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3621-3627
Number of pages7
JournalBiochemistry
Volume30
Issue number15
DOIs
StatePublished - Apr 1 1991

ASJC Scopus subject areas

  • Biochemistry

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