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PDBsum entry 1mff
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Mechanism of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor: properties of the p1g, P1a, Y95f, And n97a mutants.
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Authors
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S.L.Stamps,
A.B.Taylor,
S.C.Wang,
M.L.Hackert,
C.P.Whitman.
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Ref.
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Biochemistry, 2000,
39,
9671-9678.
[DOI no: ]
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PubMed id
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Abstract
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Phenylpyruvate tautomerase (PPT) has been studied periodically since its
activity was first described over forty years ago. In the last two years, the
mechanism of PPT has been investigated more extensively because of the discovery
that PPT is the same protein as the immunoregulatory cytokine known as
macrophage migration inhibitory factor (MIF). The mechanism of PPT is likely to
involve general base-general acid catalysis. While several lines of evidence
implicate Pro-1 as the general base, the identity of the general acid remains
unknown. Crystal structures of MIF with the competitive inhibitor
(E)-2-fluoro-p-hydroxycinnamate bound in the active site and that of the protein
complexed with the enol form of a substrate, (p-hydroxyphenyl)pyruvate, suggest
that Tyr-95 is the only candidate in the vicinity that can function as a general
acid catalyst. Although Tyr-95 is nearby the bound inhibitor and substrate, it
is not within hydrogen bonding distance of either ligand. In this study, Tyr-95
was mutated to phenylalanine, and the kinetic and structural properties of the
Y95F mutant were determined. This alteration produces a fully active enzyme,
which shows no significant structural changes in the active site. The results
indicate that Tyr-95 does not function as the general acid catalyst in the
reaction catalyzed by wild-type PPT. The mechanism of PPT was studied further by
constructing and characterizing the kinetic properties of two mutants of Pro-1
(P1G and P1A) and one mutant of Asn-97 (N97A). The mutation of Asn-97, a residue
implicated in the binding of the phenolic hydroxy group of the keto and enol
isomers of (p-hydroxyphenyl)pyruvate and of (E)-2-fluoro-p-hydroxycinnamate
affects only the binding affinity of the inhibitor. However, the mutations of
Pro-1 have a profound effect on the values of k(cat) and k(cat)/K(m) and clearly
show that Pro-1 is a critical residue in the reaction. The results are discussed
in terms of a mechanism in which Pro-1 functions as both the general acid and
the general base catalyst.
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