Tuning the activity of Cu,Zn superoxide dismutase through site-directed mutagenesis: a relatively active monomeric species |
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| Authors: | Lucia Banci I. Bertini Maria Silvia Viezzoli Emanuele Argese Emilio F. Orsega Choi Ying Chiu Guy T. Mullenbach |
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| Affiliation: | (1) Department of Chemistry, University of Florence, Via Gino Capponi 7, I-50121 Florence, Italy Tel.: +39-55-2747549; Fax: +39-55-2757555, IT;(2) Department of Environmental Sciences, University of Venice, Venice, Italy, IT;(3) Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608, USA, US;(4) Indus Biomolecular Group, 2840 College Avenue, Suite H, Berkeley, CA 94705, USA, US |
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| Abstract: | An enzymatically active monomeric analog of human copper,zinc superoxide dismutase (SOD) was produced by replacing four hydrophobic residues at the dimer interface of wild-type SOD (WT) with hydrophilic residues in a manner which has maintained the overall protein charge (i.e., Phe50Glu, Gly51Glu, Val148Lys, Ile151Lys). This analog has been characterized by (1) molecular weight determination, (2) several spectroscopic techniques probing catalytic site geometry and (3) enzymatic activity measurements at various ionic strengths. At physiological ionic strength the present monomer has sizable activity being five times that of a previously reported monomeric analog carrying only two of these substitutions with an overall charge two units more negative than WT (i.e., Phe50Glu, Gly51Glu). Unlike the catalytic activity of the latter analog, this one reveals an ionic strength dependency like that of WT. Enzymatic behavior is discussed with regard to factors affecting substrate diffusion towards the catalytic site. Received: 11 October 1996 / Accepted: 24 February 1997 |
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| Keywords: | Copper zinc superoxide dismutase Monomeric mutant Enzymatic activity Ionic strength Spectroscopic investigation |
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