Mechanistic implications of methylglyoxal synthase complexed with phosphoglycolohydroxamic acid as observed by X-ray crystallography and NMR spectroscopy

Methylglyoxal synthase (MGS) and triosephosphate isomerase (TIM) share neit her sequence nor structural similarities, yet the reactions catalyzed by bo th enzymes are similar, in that both initially convert dihydroxyacetone pho sphate to a cis-enediolic intermediate. This enediolic intermediate is form ed 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 elimi nation of phosphate from this enediolic intermediate to form the enol of me thylglyoxal, while TIM catalyzes proton donation to C2 to form D-glyceralde hyde phosphate. A competitive inhibitor of TIM, phosphoglycolohydroxamic ac id (PGH) is found to be a tight binding competitive inhibitor of MGS with a Ki of 39 nM. PGH's high affinity for MGS may be due in part to a short, st rong hydrogen bond (SSHB) from the NOH of PGH to the carboxylate of Asp-71. Evidence for this SSHB is found in X-ray, LH NMR, and fractionation factor data. The X-ray structure of the MGS homohexamer complexed with PGH at 2.0 Angstrom resolution shows this distance to be 2.30-2.37 +/- 0.24 Angstrom. H-1 NMR shows a PGH-dependent 18.1 ppm signal that is consistent with a hy drogen bond length of 2.49 +/- 0.02 Angstrom. The D/H fractionation factor (phi = 0.43 coproduct 0.02) is consistent with a hydrogen bond length of 2. 53 +/- 0.01 Angstrom. Further, N-15 NMR suggests a significant partial posi tive charge on the nitrogen atom of bound PGH, which could strengthen hydro gen bond donation to Asp-71. Both His-98 and His-19 are uncharged in the MG S-PGH complex on the basis of the chemical shifts of their C delta and C ep silon 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 fr om 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 a nd 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 ini tially abstracts the C3 pro-S proton from DHAP and subsequently the 3-OH pr oton from the enediolic intermediate. This mechanism is compared to an alte rnative TIM-like mechanism for MGS, and the relative merits of both mechani sms are discussed.