Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase  Michael J. Reddish, Robert Callender, R. Brian Dyer  Biophysical Journal  Volume 112, Issue 9, Pages 1852-1862 (May 2017) DOI: 10.1016/j.bpj.2017.03.031 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Structure of the pig heart LDH active site (PDB:5LDH). Most of the protein is colored gray as a cartoon diagram or hidden for clarity. Key active site residues are highlighted as stick structures in other colors with their bonding interactions highlighted as black dashed lines. These interactions include Arg-109 on the mobile loop (green) and Arg-171 and His-195 (orange). The crystallized S-Lac-NAD+ substrate-cofactor analog has been colored to represent the NAD+ cofactor segment in cyan and the lactate segment in magenta; to emphasize each component, the bond between the cofactor and substrate mimic segments is not displayed. Image created with PyMOL (www.pymol.org) (38). To see this figure in color, go online. Biophysical Journal 2017 112, 1852-1862DOI: (10.1016/j.bpj.2017.03.031) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Equilibrium fluorescence of LDH⋅NADH complexes. The complexes are LDH⋅NADH with no substrate or substrate analog (black); LDH⋅NAD+⋅lactate (blue) produced by mixing LDH⋅NADH and pyruvate; LDH⋅NADH⋅oxamate (red); and a reference NADH solution (green, dashed). (A) Spectra excited at 280 nm showing tryptophan emission centered around 350 nm and FRET from tryptophan to NADH centered around 445 nm. (B) Spectra excited at 340 nm showing NADH emission centered around 445 nm. To see this figure in color, go online. Biophysical Journal 2017 112, 1852-1862DOI: (10.1016/j.bpj.2017.03.031) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Tryptophan-probed mixing transient of LDH⋅NADH and pyruvate or oxamate. The data are presented as the normalized fluorescence intensity of tryptophan emission (excitation: 280 nm, emission: 340 nm). Single-exponential fits to the data are shown and their fit rates are listed in the legend. The fit shown for the oxamate data is shown to emphasize the difference in the traces, but should not be taken as an accurate fit because the fit lifetime is outside the observation window. To see this figure in color, go online. Biophysical Journal 2017 112, 1852-1862DOI: (10.1016/j.bpj.2017.03.031) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 FRET-probed mixing transient of LDH⋅NADH and pyruvate or oxamate. The data are presented as the normalized fluorescence intensity of FRET emission, tryptophan to NADH (excitation: 280 nm, emission: 460 nm). Exponential fits to the data are shown; see text. To see this figure in color, go online. Biophysical Journal 2017 112, 1852-1862DOI: (10.1016/j.bpj.2017.03.031) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 NADH-probed mixing transient of LDH⋅NADH and pyruvate or oxamate. The data are presented as the normalized fluorescence intensity of NADH emission (excitation: 355 nm, emission: 460 nm). Fits to the data are shown and their representative fit rates are listed in the legend. See the text for a discussion of the fitting functions used. To see this figure in color, go online. Biophysical Journal 2017 112, 1852-1862DOI: (10.1016/j.bpj.2017.03.031) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Absorption (A) and fluorescence (B) T-jump kinetics of the reaction system: 0.4 mN LDH, 0.4 mM NAD+, and 5 mM lactate (total initial concentrations) to a final temperature of 22°C. Transients are also shown where C2-d-lactate was used to measure the primary kinetic isotope effect. After irradiation with the 20 ns laser pulse induces the T-jump, the temperature in the sample remains constant until the millisecond time range; the temperature decays to ambient with a 30 ms half-time. Hence the lifetimes of any observed kinetics may contain a strong cooling component past 10 ms. Double-exponential fits between 1 μs and 10 ms are shown in the plots as black lines, and their fit rates are given in the legend. The ragged features in (A) below 1 μs are artifacts, due to cavitation effects. To see this figure in color, go online. Biophysical Journal 2017 112, 1852-1862DOI: (10.1016/j.bpj.2017.03.031) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 Comparison of the T-jump absorption kinetics of the LDH reaction system when probed by the infrared absorbance of the C-2 pyruvate carbonyl stretch at 1685 cm−1 and when probed by the NADH absorbance at 340 nm. The infrared data is from http://pubs.acs.org/doi/abs/10.1021/jp5050546 (15), reproduced by permission. The UV data is under the same conditions as Fig. 6, except the T-jump is from 24 to 32°C to more closely match the infrared conditions. The bumps near 5 μs are artifacts due to cavitation effects. To see this figure in color, go online. Biophysical Journal 2017 112, 1852-1862DOI: (10.1016/j.bpj.2017.03.031) Copyright © 2017 Biophysical Society Terms and Conditions