High‐Precision Measurement of Hydrogen Bond Lengths in Proteins by Nuclear Magnetic Resonance Methods

We have compared hydrogen bond lengths on enzymes derived with high precision (≤ ±0.05 Å) from both the proton chemical shifts (δ) and the fractionation factors (ϕ) of the proton involved with those obtained from protein X‐ray crystallography. Hydrogen bond distances derived from proton chemical shifts were obtained from a correlation of 59 O—H····O hydrogen bond lengths, measured by small molecule high‐resolution X‐ray crystallography, with chemical shifts determined by solid‐state nuclear magnetic resonance (NMR) in the same crystals (McDermott A, Ridenour CF, Encyclopedia of NMR, Sussex, U.K.: Wiley, 1996:3820–3825). Hydrogen bond distances were independently obtained from fractionation factors that yield distances between the two proton wells in quartic double minimum potential functions (Kreevoy MM, Liang TM, J Am Chem Soc, 1980;102:3315–3322). The high‐precision hydrogen bond distances derived from their corresponding NMR‐measured proton chemical shifts and fractionation factors agree well with each other and with those reported in protein X‐ray structures within the larger errors (±0.2–0.8 Å) in distances obtained by protein X‐ray crystallography. The increased precision in measurements of hydrogen bond lengths by NMR has provided insight into the contributions of short, strong hydrogen bonds to catalysis for several enzymatic reactions. Proteins 1999;35:275–282. © 1999 Wiley‐Liss, Inc

NMR Studies of the Role of Hydrogen Bonding in the Mechanism of Triosephosphate Isomerase

Triosephosphate isomerase (TIM) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP), with Glu-165 removing the pro-R proton from C1 of DHAP and neutral His-95 polarizing the carbonyl group of the substrate. TIM and its complexes with the reactive intermediate analogs, phosphoglycolic acid (PGA) and phosphoglycolohydroxamic acid (PGH), were studied by 1H NMR at 600 MHz and at low temperature (−4.8 °C). His-95 shows an NεH resonance at 13.1 ppm which shifts to 13.3 ppm in the TIM−PGA complex and to 13.5 ppm in the TIM−PGH complex. In the TIM−PGH complex, His-95 NεH shows a slow, pH-independent exchange rate with water (k ex = 80 s-1 at 30 °C, E act = 19 kcal/mol), which is 44-fold slower than that of an exposed histidine suggesting partial shielding from bulk solvent, and a fractionation factor φ = 0.71 ± 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 ppm is absent and the resonance at 10.9 ppm is much weaker in the TIM complex of PGA, which lacks the hydroxamic acid (−NHOH) moiety. 15N-labeled PGH was synthesized and the NH proton of free [15N]PGH shows a single 1H−15N HMQC cross peak with δ(1H) = 10.3 ppm and δ(15N) = 168 ppm which shifts to δ(1H) = 10.9 ppm and δ(15N) = 174 ppm in the TIM complex of [15N]PGH. The 15N−1H coupling in the complex indicates covalent N−H bonding, and the deshielded δ(15N) indicates a significant contribution of the imidate resonance form of PGH. The 14.9 ppm resonance is assigned to the NOH proton of bound PGH. This resonance shows a pH-independent exchange rate with water (k ex = 3900 s-1 at 30 °C, E act = 8.9 kcal/mol) which may reflect 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 acetohydroxamic acid, and a very low fractionation factor φ = 0.38 ± 0.06. In 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 bonded to Glu-165. A low-barrier hydrogen bond from PGH NOH to Glu-165 suggests 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 in the interconversion of DHAP and GAP in wild type TIM. Such a mechanism, together with the measured exchange rate of the His-95 NεH proton with solvent protons can accommodate the classical measurements of tritium incorporation from DHAP into GAP

Vaccinia DNA Topoisomerase I: Evidence Supporting a Free Rotation Mechanism for DNA Supercoil Relaxation

The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA supercoils. For type I topoisomerases, two mechanisms have been proposed for supercoil release:  (1) a coupled mechanism termed strand passage, in which a single supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s-1), religation (4 s-1), and the cleavage equilibrium constant on the enzyme (0.075) at 22 °C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (∼20 rotations/s). Thus, the average number of supercoils removed for each cleavage event greatly exceeds unity (Δn = 5) and depends on kinetic competition between religation and supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes