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Active Site Properties of [Aspartic acid 43]-Semisynthetic Nuclease-T
Journal article   Open access   Peer reviewed

Active Site Properties of [Aspartic acid 43]-Semisynthetic Nuclease-T

Irwin M. Chaiken and Guillermo R. Sánchez
The Journal of biological chemistry, v 247(21), pp 6743-6747
Nov 1972
DOI: https://doi.org/10.1016/S0021-9258(19)44649-8
PMID: 4343156
url
https://doi.org/10.1016/s0021-9258(19)44649-8View
Published, Version of Record (VoR) Open
url
https://doi.org/10.1016/S0021-9258(19)44649-8View
Published, Version of Record (VoR) Open

Abstract

[Aspartic acid 43]-semisynthetic staphylococcal nuclease-T′ is an active site analogue of native nuclease-T′, the enzymically active proteolytic derivative of staphylococcal nuclease containing the noncovalently bound fragments nuclease-T-(6–48) (residues 6 through 48 of nuclease) and nuclease-T-(49–149) (residues 49 through 149). The semisynthetic analogue complex, which contains [aspartic acid 43]-synthetic-(6–47) in place of nuclease-T-(6–48), has been obtained by functional purification. This complex has an over-all folded structure similar to that of normal semisynthetic nuclease-T′ but lacks enzymic activity. Nonetheless, [aspartic acid 43]-semisynthetic nuclease-T′ binds the active site ligands deoxythymidine-3′,5′-diphosphate (an inhibitor of nuclease and nuclease-T′) and Ca++ (an ion essential for nuclease and nuclease-T′ activity). Binding of ligands is interdependent, a property characteristic for native nuclease-T′, but appears somewhat weaker than that for the native complex. Thus, replacement of glutamic acid 43 by aspartic acid causes limited but important steric changes in the active site region leading to the direct perturbation of the catalytic process. Based on these results, together with the prior knowledge that glutamic acid 43 is in the active site region in the crystal structure of nuclease and that nuclease and nuclease-T′ have similar folded structures, it is concluded that glutamic acid 43 is critical for the proper organization of the active site in nuclease and nuclease-T′.

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