1ik4
|
X-ray Structure of Methylglyoxal Synthase from E. coli Complexed with Phosphoglycolohydroxamic Acid
OverviewOverview
Methylglyoxal synthase (MGS) and triosephosphate isomerase (TIM) share, neither sequence nor structural similarities, yet the reactions catalyzed, by both enzymes are similar, in that both initially convert, dihydroxyacetone phosphate to a cis-enediolic intermediate. This enediolic, intermediate is formed from the abstraction of the pro-S C3 proton of DHAP, by Asp-71 of MGS or the pro-R C3 proton of DHAP by Glu-165 of TIM. MGS, then catalyzes the elimination of phosphate from this enediolic, intermediate to form the enol of methylglyoxal, while TIM catalyzes proton, donation to C2 to form D-glyceraldehyde phosphate. A competitive inhibitor, of TIM, phosphoglycolohydroxamic acid (PGH) is found to be a tight binding, competitive inhibitor of MGS with a K(i) of 39 nM. PGH's high affinity for, MGS may be due in part to a short, strong hydrogen bond (SSHB) from the, NOH of PGH to the carboxylate of Asp-71. Evidence for this SSHB is found, in X-ray, 1H NMR, and fractionation factor data. The X-ray structure of, the MGS homohexamer complexed with PGH at 2.0 A resolution shows this, distance to be 2.30-2.37 +/- 0.24 A. 1H NMR shows a PGH-dependent 18.1 ppm, signal that is consistent with a hydrogen bond length of 2.49 +/- 0.02 A., The D/H fractionation factor (phi = 0.43 +/- 0.02) is consistent with a, hydrogen bond length of 2.53 +/- 0.01 A. Further, 15N NMR suggests a, significant partial positive charge on the nitrogen atom of bound PGH, which could strengthen hydrogen bond donation to Asp-71. Both His-98 and, His-19 are uncharged in the MGS-PGH complex on the basis of the chemical, shifts of their Cdelta and C(epsilon) protons. The crystal structure, reveals that Asp-71, on the re face of PGH, and His-19, on the si face of, PGH, both approach the NO group of the analogue, while His-98, in the, plane of PGH, approaches the carbonyl oxygen of the analogue. The, phosphate group of PGH accepts nine hydrogen bonds from seven residues and, is tilted out of the imidate plane of PGH toward the re face. Asp-71 and, phosphate are thus positioned to function as the base and leaving group, respectively, in a concerted suprafacial 1,4-elimination of phosphate from, the enediolic intermediate in the second step of the MGS reaction., Combined, these data suggest that Asp-71 is the one base that initially, abstracts the C3 pro-S proton from DHAP and subsequently the 3-OH proton, from the enediolic intermediate. This mechanism is compared to an, alternative TIM-like mechanism for MGS, and the relative merits of both, mechanisms are discussed.
About this StructureAbout this Structure
1IK4 is a Single protein structure of sequence from Escherichia coli with PGH as ligand. Active as Methylglyoxal synthase, with EC number 4.2.3.3 Full crystallographic information is available from OCA.
ReferenceReference
Mechanistic implications of methylglyoxal synthase complexed with phosphoglycolohydroxamic acid as observed by X-ray crystallography and NMR spectroscopy., Marks GT, Harris TK, Massiah MA, Mildvan AS, Harrison DH, Biochemistry. 2001 Jun 12;40(23):6805-18. PMID:11389594
Page seeded by OCA on Tue Nov 20 17:27:47 2007