Abstract:
:3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P, a key precursor in the biosynthesis of the endotoxin of Gram-negative bacteria. Earlier studies have established that the condensation occurs with a syn addition of water to the si side of C2(PEP) and of C3(PEP) to the re side of C1(A5P). Two stepwise mechanisms have been proposed for this reaction. One involves a transient carbanion intermediate, formed by attack of water or a hydroxide ion on C2(PEP). The other involves a transient oxocarbenium zwitterionic intermediate, formed by direct attack of C3(PEP) onto C1(A5P), followed by reaction of water at C2. In both cases, the transient intermediates are expected to converge to a more stable tetrahedral intermediate, which decays into KDO8P and inorganic phosphate. In this study we calculated the potential energy surfaces (PESs) associated with all possible reaction paths in the active site of KDO8PS: the path involving a syn addition of water to the si side of C2(PEP) and of C3(PEP) to the re side of C1(A5P), with the PEP phosphate group deprotonated, has the lowest energy barrier ( approximately 14 kcal/mol) and is strongly exoergonic (reaction energy of -38 kcal/mol). Consistent with the experimental observations, other potential reaction paths, like an anti addition of water to the re side of C2(PEP) or addition of C3(PEP) to the si side of C1(A5P), are associated with much higher barriers. An important new finding of this study is that the lowest energy reaction path does not correspond to either one of the pure stepwise mechanisms proposed formerly but can be described instead as a partially concerted reaction between PEP, A5P, and water. The success in using PESs to reproduce established features of the reaction and to discriminate between different mechanisms suggests that this approach may be of general utility in the study of other enzymatic reactions.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Tao P,Gatti DL,Schlegel HBdoi
10.1021/bi901341hsubject
Has Abstractpub_date
2009-12-15 00:00:00pages
11706-14issue
49eissn
0006-2960issn
1520-4995journal_volume
48pub_type
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