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Title
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Structure of isocitrate dehydrogenase with isocitrate, nicotinamide adenine dinucleotide phosphate, and calcium at 2.5-A resolution: a pseudo-Michaelis ternary complex.
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Authors
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B.L.Stoddard,
A.Dean,
D.E.Koshland.
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Ref.
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Biochemistry, 1993,
32,
9310-9316.
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PubMed id
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Abstract
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The structure of isocitrate dehydrogenase (IDH) with a bound complex of
isocitrate, NADP+, and Ca2+ was solved at 2.5-A resolution and compared by
difference mapping against previously determined enzymatic complexes. Calcium
replaces magnesium in the binding of metal-substrate chelate complex, resulting
in a substantially reduced turnover rate. The structure shows the following: (i)
A complete, structurally ordered ternary complex (enzyme, isocitrate, NADP+, and
Ca2+) is observed in the active site, with the nicotinamide ring of NADP+
exhibiting a specific salt bridge with isocitrate. The binding of the cofactor
nicotinamide ring is dependent on this interaction. (ii) Isocitrate is bound by
the enzyme with the same interactions as those found for the magnesium/substrate
binary complex, but the entire molecule is shifted in the active site by
approximately 1 A in order to accommodate the larger metal species and to
interact with the nicotinamide ring. The distances from isocitrate to the bound
calcium are substantially longer than those previously found with magnesium.
(iii) NADP in the Escherichia coli IDH has a novel binding site and conformation
as compared to previously solved dehydrogenases. (iv) The orientation and
interactions of the nicotinamide ring with the substrate are consistent with the
stereospecificity of the enzyme-catalyzed reaction.
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