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)].(ABSTRACT TRUNCATED AT 250 WORDS)
Poole
L B
LB
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Loveys
D A
DA
Hale
S P
SP
Gerlt
J A
JA
Stanczyk
S M
SM
Bolton
P H
PH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014961:X-Ray Diffraction
D008836:Micrococcal Nuclease; D002118:Calcium