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PDBsum entry 2a0x
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References listed in PDB file
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Key reference
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Title
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Neighboring group participation in the transition state of human purine nucleoside phosphorylase.
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Authors
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A.S.Murkin,
M.R.Birck,
A.Rinaldo-Matthis,
W.Shi,
E.A.Taylor,
S.C.Almo,
V.L.Schramm.
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Ref.
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Biochemistry, 2007,
46,
5038-5049.
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PubMed id
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Abstract
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The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with
bound inosine or transition-state analogues show His257 within hydrogen bonding
distance of the 5'-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp
exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic
of an early transition state as much as 370-fold (Km/Ki) less tightly than
native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late
transition state, nearly as well as the native enzyme. These results indicate
that His257 serves an important role in the early stages of transition-state
formation. Whereas mutation of His257 resulted in little variation in the PNP x
DADMe-ImmH x SO4 structures, His257Phe x ImmH x PO4 showed distortion at the
5'-hydroxyl, indicating the importance of H-bonding in positioning this group
during progression to the transition state. Binding isotope effect (BIE) and
kinetic isotope effect (KIE) studies of the remote 5'-(3)H for the arsenolysis
of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large
intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic
distortion toward the transition state in the Michaelis complex with continued
development of a similar distortion at the transition state. The mutants
His257Phe, His257Gly, and His257Asp altered the 5'-(3)H intrinsic KIE to -3,
-14, and 7%, respectively, while the BIEs contributed 2, 2, and -2%,
respectively. These surprising results establish that forces in the Michaelis
complex, reported by the BIEs, can be reversed or enhanced at the transition
state.
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