1jci
From Proteopedia
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Stabilization of the Engineered Cation-binding Loop in Cytochrome c Peroxidase (CcP)
Overview
We have previously shown that the K(+) site found in the proximal heme, pocket of ascorbate peroxidase (APX) could be successfully engineered into, the closely homologous cytochrome c peroxidase (CcP) [Bonagura et al., (1996) Biochemistry 35, 6107-6115; Bonagura et al. (1999) Biochemistry 38, 5538-5545]. In addition, specificity could be switched to binding Ca(2+), as found in other peroxidases [Bonagura et al. (1999) J. Biol. Chem. 274, 37827-37833]. The introduction of a proximal cation-binding site also, promotes conversion of the Trp191 containing cation-binding loop from a, "closed" to an "open" conformer. In the present study we have changed a, crucial hinge residue of the cation-binding loop, Asn195, to Pro which, stabilizes the loop, albeit, only in the presence of bound K(+). The, crystal structure of this mutant, N195PK2, has been refined to 1.9 A. As, predicted, introduction of this crucial hinge residue stabilizes the, cation-binding loop in the presence of the bound K(+). As in earlier work, the characteristic EPR signal of Trp191 cation radical becomes, progressively weaker with increasing [K(+)] and the lifetime of the Trp191, radical also has been considerably shortened in this mutant. This mutant, CcP exhibits reduced enzyme activity, which could be titrated to lower, levels with increasing [K(+)] when horse heart cytochrome c is the, substrate. However, with yeast cytochrome c as the substrate, the mutant, was as active as wild-type at low ionic strength, but 40-fold lower at, high ionic strength. We attribute this difference to a change in the, rate-limiting step as a function of ionic strength when yeast cytochrome c, is the substrate.
About this Structure
1JCI is a Single protein structure of sequence from Saccharomyces cerevisiae with K and HEM as ligands. Active as Cytochrome-c peroxidase, with EC number 1.11.1.5 Full crystallographic information is available from OCA.
Reference
Cation-induced stabilization of the engineered cation-binding loop in cytochrome c peroxidase (CcP)., Bhaskar B, Bonagura CA, Li H, Poulos TL, Biochemistry. 2002 Feb 26;41(8):2684-93. PMID:11851415
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