NMR-STUDIES OF THE ROLE OF HYDROGEN-BONDING IN THE MECHANISM OF TRIOSEPHOSPHATE ISOMERASE

Triosephosphate isomerase (TIM) catalyzes the reversible interconversi on of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP), with Glu-165 removing the pro-R proton from C1 of DHAP and neu tral His-95 polarizing the carbonyl group of the substrate. TIM and it s complexes with the reactive intermediate analogs, phosphoglycolic ac id (PGA) and phosphoglycolohydroxamic acid (PGH), were studied by H-1 NMR at 600 MHz and at low temperature (-4.8 degrees C). His-95 shows a n N epsilon H resonance at 13.1 ppm which shifts to 13.3 ppm in the TI M-PGA complex and to 13.5 ppm in the TIM-PGH complex. In the TIM-PGH c omplex, His-95 N epsilon H shows a slow, pH-independent exchange rate with water (k(ex) = 80 s(-1) at 30 degrees C, E-act = 19 kcal/mol), wh ich is 44-fold slower than that of an exposed histidine suggesting par tial shielding from bulk solvent, and a fractionation factor phi = 0.7 1 +/- 0.02 consistent with its donation of a normal hydrogen bond. The formation of the TIM-PGH complex results in the appearance of several deshielded proton resonances, including one at 14.9 ppm and one at 10 .9 ppm which overlaps with another resonance. The resonance at 14.9 pp m is absent and the resonance at 10.9 ppm is much weaker in the TIM co mplex of PGA, which lacks the hydroxamic acid (-NHOH) moiety. N-15-lab eled PGH was synthesized and the NH proton of free [N-15]PGH shows a s ingle H-1-N-15 HMQC cross peak with delta(H-1) = 10.3 ppm and delta(N- 15) = 168 ppm which shifts to delta(H-1) = 10.9 ppm and delta(N-15) = 174 ppm in the TIM complex of [N-15]PGH. The N-15-H-1 coupling in the complex indicates covalent N-H bonding, and the deshielded delta(N-15) indicates a significant contribution of the imidate resonance form of PGH. The 14.9 ppm resonance is assigned to the NOH proton of bound PG H. This resonance shows a pH-independent exchange rate with water (k(e x) = 3900 s(-1) at 30 degrees C, E-act = 8.9 kcal/mol) which may refle ct the dissociation of the TIM-PGH complex, and meets the criteria for a low-barrier hydrogen bond on the basis of the significant downfield shift of 6.2 ppm from the NOH proton of the model compound acetohydro xamic acid, and a very low fractionation factor phi = 0.38 +/- 0.06. I n the X-ray structure of the TIM-PGH complex [Davenport, R. C., Bash, P. A., Seaton, B. A., Karplus, M., Petsko, G. A., and Ringe, D. (1991) Biochemistry 30, 5821], the NOH proton of bound PGH is hydrogen bonde d to Glu-165. A low-barrier hydrogen bond from PGH NOH to Glu-165 sugg ests a dual role for Glu-165 in catalysis of proton transfer not only between the C1 and C2 carbons but also between the O1 and O2 oxygens i n the interconversion of DHAP and GAP in wild type TIM. Such a mechani sm, together with the measured exchange rate of the His-95 N epsilon H proton with solvent protons can accommodate the classical measurement s of tritium incorporation from DHAP into GAP.