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Design of a folded, conformationally stable oxaloacetate decarboxylase

Taylor, Susan E., Rutherford, Trevor J. and Allemann, Rudolf Konrad 2002. Design of a folded, conformationally stable oxaloacetate decarboxylase. Journal of the Chemical Society, Perkin Transactions 2 2 (4) , pp. 751-755. 10.1039/b200057c

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Abstract

Oxaldie-4, a 31-residue polypeptide designed to catalyse the decarboxylation of oxaloacetate, has been synthesised and its structural and catalytic properties characterised. The solution structure of Oxaldie-4 was studied by CD and NMR spectroscopy. Oxaldie-4, the design of which was based on bovine pancreatic polypeptide, adopted a stably folded structure in solution, which was characterised by the tight packing of a poly-proline-like helix and an α-helix as shown by a large number of inter-helix NOEs. The structure of Oxaldie-4 was in sharp contrast to the molten globule-like structure formed by Oxaldie-3, which was based on avian pancreatic polypeptide. The stability of Oxaldie-4 with respect to thermal and urea denaturation was significantly improved when compared to Oxaldie-3. Oxaldie-4 catalysed the decarboxylation of oxaloacetate with Michaelis-Menten saturation kinetics. The kinetic parameters, which were independent of the concentration of the catalyst over the whole range studied, were determined in a spectrophotometric assay at pH 7 and 298 K to be 0.229 s−1, 64.8 mM, and 2.9 M−1 s−1 for kcat, KM, and kcat/KM, respectively. This catalytic efficiency corresponds to a rate increase of almost four orders of magnitude when compared to simple amines such as butylamine. However, despite the stable three-dimensional structure, the catalytic efficiency of Oxaldie-4 was only slightly improved relative to Oxaldie-3, most likely the consequence of the high flexibility of the lysine side chains, which make up the active site of Oxaldie-4.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Cardiff Catalysis Institute (CCI)
Subjects: Q Science > QD Chemistry
Publisher: Royal Society of Chemistry
ISSN: 1472-779X
Last Modified: 04 Jun 2017 03:04
URI: http://orca.cf.ac.uk/id/eprint/16232

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