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OwnerGerard W. Canters, Hein Jakob Wijma +2inventors
InventorsGerard W. Canters, Hein Jakob Wijma, Michael Murphy, Iain Macpherson
Estimating the term…
In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type 1 site was replaced (M150G) to make the copper atom accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A460/A600=0.71) differs significantly from that of the native nitrite reductase (A460/A600=1.3). The reduction potential of the type-1 site of nitrite reductase M150G (EM=312−5 mV versus hydrogen) is higher than that of the native enzyme (EM=213−5 mV). M150G has a lower catalytic activity (kcat=133−6 s−1) than the wild-type nitrite reductase (kcat=416−10−s 1). The binding of external ligands to M150G restores spectral properties, reduction potential (EM<225 mV), and catalytic activity (kcat=374−28 s−1). Also the M150H (A460/A600=7.7, EM=104−5 mV, kcat=0.099−0.006 s−1) and M150T (A460/A600=0.085, EM=340−5 mV, kcat=126−2 s−1) variants were characterized to compare their properties with those of M150G. Crystal structures show that the ligands act as allosteric effectors by displacing Met62 which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that a rearranged ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.
A method of detecting redox enzyme activity in which an electron transfer enzyme derived from a wild type oxidoreductase having a type-1 copper site is contacted with a substrate for the enzyme to oxidise or reduce the substrate and the enzyme activity is monitored via the activity of an oxidant or reductant, as the case may be, of the type 1 copper site, characterised in that the type 1 copper site has been modified compared to the wild type enzyme by substitution of a copper coordinating residue which coordinates the copper ion of the type 1 site by a residue selected from Gly and Ala, and the enzymatic reaction is carried out in the presence of an allosteric effector, which is a solute molecule which is capable of modifying the activity of the enzyme to allow an electron donating residue of the enzyme to coordinate with the copper ion of the type 1 copper site.
A redox enzyme comprising at least one copper ion and comprising sequence ID1 in which one of the residues His95, Cys136 and Met150 is substituted by a residue selected from Gly and Ala and in which the other of such residues is conserved, in which Met62 is conserved, and in which the remaining residues are identical or up to 50% of them may be conservatively substituted, and/or in which up to 10 residues at the C and/or N terminal of the SEQ ID NO:1 are deleted.
A sensor comprising an electrode and, in contact with the electrode, a reaction medium containing:
(canceled)
Dependencies are inferred from the claim text.