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A Solid Phase Synthetic Study of the Active Site Region of Staphylococcal Nuclease-T
Journal article   Open access   Peer reviewed

A Solid Phase Synthetic Study of the Active Site Region of Staphylococcal Nuclease-T

Irwin M. Chaiken and Christian B. Anfinsen
The Journal of biological chemistry, v 246(7), pp 2285-2290
10 Apr 1971
DOI: https://doi.org/10.1016/S0021-9258(19)77219-6
PMID: 5576073
url
https://doi.org/10.1016/s0021-9258(19)77219-6View
Published, Version of Record (VoR) Open
url
https://doi.org/10.1016/S0021-9258(19)77219-6View
Published, Version of Record (VoR) Open

Abstract

Solid phase synthetic peptides have been prepared corresponding to the sequence in staphylococcal nuclease of residues 6 through 47, as well as single substitution analogues of this region in which aspartic acid 21, arginine 35, aspartic acid 40, or glutamic acid 43 was replaced with a closely related residue. Aspartic acid was replaced with either asparagine or glutamic acid, arginine with either citrulline or lysine, and glutamic acid with aspartic acid. All synthetic peptides were examined for the ability to form an enzymically active complex analogous to the nuclease-T' complex formed by nuclease-T-(6-48) (nuclease residues 6 through 48) with the native fragment nuclease-T-(49-149) (nuclease residues 49 or 50 through 149). Whereas [Asn40]-synthetic-(6-47) formed a complex with nuclease-T-(49-149) with about 25% the activity of that formed with the normal synthetic peptide, synthetic-(6-47), none of the remaining analogues generated activity. All four "charge-preserved" analogues, namely [Glu21]-, [Lys35]-, [Glu40]-, and [Asp43]-synthetic-(6-47), formed inactive complexes with nuclease-T-(49-149), while the "amide" analogues [Asn21]- and [Cit35]-synthetic-(6-47) did not bind to the native fragment effectively. These, together with earlier data on an analogue with glutamine rather than glutamic acid at position 43 (Ontjes, D. A., and Anfinsen, C. B., J. Biol. Chem., 244, 6316 (1969)), indicate the importance of all of residues 21, 35, 40, and 43 in the formation of enzymically active nuclease-T'. The results support the view, suggested by the structure of nuclease deduced from x-ray studies, that these 4 residues play critical roles in the binding of competitive inhibitors and calcium ions.

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