Stewart N. Loh

The Energetics of Hydrogen Bonds in Model Systems: Implications for Enzymatic Catalysis

Low-barrier or short, strong hydrogen bonds have been proposed to contribute 10 to 20 kilocalories per mole to transition-state stabilization in enzymatic catalysis. The proposal invokes a large increase in hydrogen bond energy when the pKa values of the donor and acceptor (where Ka is the acid constant) become matched in the transition state (ΔpKa = 0). This hypothesis was tested by investigating the energetics of hydrogen bonds as a function of ΔpKa for homologous series of compounds under nonaqueous conditions that are conducive to the formation of low-barrier hydrogen bonds. In all cases, there was a linear correlation between the increase in hydrogen-bond energy and the decrease in ΔpKa, as expected from simple electrostatic effects. However, no additional energetic contribution to the hydrogen bond was observed at ΔpKa = 0. These results and those of other model studies suggest alternative mechanisms by which hydrogen bonds can contribute to enzymatic catalysis, in accord with conventional electrostatic considerations.

Thermodynamic origin of prolyl peptide bond isomers

The thermodynamic preference for the trans isomer of prolyl peptide bonds arises almost entirely from enthalpy in aqueous buffer and in toluene.

Measurement of Amide Hydrogen D/H Fractionation Factors in Proteins by NMR Spectroscopy

Publisher Summary This chapter discusses the measurement of amide hydrogen D/H fractionation factors in proteins by nuclear magnetic resonance (NMR) spectroscopy. It presents a new method that employs two-dimensional heteronuclear NMR spectroscopy of 15 N labeled proteins for measuring D/H fractionation factors at individual backbone amides. In developing this technique, the study uses as a model system the enzyme staphylococcal nuclease H124L (16.8 kD) labeled uniformly with 15 N to 99 atom%. Nuclease H124L (Nase) is the recombinant protein produced from Escherichia coli whose sequence is identical to that of the nuclease produced by the V8 strain of Staphylococcus aureus. Nase differs from the variant produced by the Foggi strain by having leucine at position 124 in place of histidine. The enzyme, which requires Ca 2+ for activity, catalyzes the degradation of DNA and RNA to mono- and oligonucleotides. Most importantly, for this study, nearly complete 15 N NMR peak assignments are available for Nase, and three high-resolution crystal structures have been solved for the Foggi strain enzyme.

Chemical exchange spectroscopy based on carbon-13 NMR. Applications to enzymology and protein folding

Abstract We explore how 13C-based two-dimensional chemical exchange spectroscopy (EXSY) can be used to investigate exchange processes that are slow on the NMR time scale. Results are shown for the mutarotase-catalyzed α →← β isomerization of [1-13C]glucose using experiments that detect carbon spins: homonuclear 13C exchange spectroscopy [13C {13C} EXSY] and heteronuclear exchange spectroscopy [13C {1H} EXSY]; and inverse experiments that select for proton spins attached to 13C: 1H− 13C single-bond correlation exchange spectroscopy [1H {13C} SBC-EXSY] and 13C-filtered 1H exchange spectroscopy [ 1 H { 1 H }− 13 C ƒ - EXSY ] . The main advantage of 13C-based exchange experiments is the simplification of complex spectra afforded by incorporation of selective labels. The inherent power of this approach is illustrated with a 1H {13C} SBC-EXSY spectrum showing the native →← denatured interconversion of [ 13 C δ1 ] Trp-staphylococcal nuclease. Certain 13C-based EXSY experiments are useful for discriminating exchange connectivities from dipole-dipole connectivities.

Measuring Global and Local Structural Free Energy Changes in Staphylococcal Nuclease by NMR-Observed Hydrogen Exchange

Publisher Summary This chapter explores the measurement of global and local structural free energy changes in staphylococcal nuclease by nuclear magnetic resonance (NMR)-observed hydrogen exchange. Hydrogen exchange has been used to estimate the free energy change of global unfolding, both in the presence and absence of the active site ligands Ca 2+ and pdTp. The studies described in the chapter establish a baseline level of stabilizing free energy conferred by ligand binding. A region exhibiting increases in protection above this baseline value has been identified. This has been attributed to inhibitor-induced perturbation of the P117 cis ⇋ trans equilibrium to the more exchange-stable cis conformation. The known properties of the cis ⇋ trans equilibrium about the K116–P117 peptide bond are consistent with it being the additional mechanism for facilitating exchange in a region of SNase. Mutational analysis has shown the position of the cis ⇋ trans equilibrium to be strongly correlated with the stability of the protein toward guanidine hydrochloride denaturation, with the trans-P117 isomeric form being much less stable than the cis. The protein containing trans-P117 is somewhat more expanded than the protein with P117 in the cis orientation.