Kinetic and thermodynamic rationale for suberoylanilide hydroxamic acid being a preferential human histone deacetylase 8 inhibitor as compared to the structurally similar ligand, trichostatin a.

Of the different hydroxamate-based histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic acid (SAHA) has been approved by the Food and Drug Administration for the treatment of T-cell lymphoma. Interestingly, a structurally similar inhibitor, trichostatin A (TSA), which has a higher in vitro inhibitory potency against HDAC8, reportedly shows poor efficacy in clinical settings. To gain molecular insight into this discriminatory feature, we performed transient kinetic and isothermal titration calorimetric studies for the interaction of SAHA and TSA with the recombinant form of human HDAC8. The transient kinetic data revealed that the binding of both inhibitors to the enzyme showed biphasic profiles, which represented an initial encounter of the enzyme with the inhibitor followed by the isomerization of the transient enzyme-inhibitor complexes. The temperature-dependent transient kinetic studies with these inhibitors revealed that the bimolecular process is primarily dominated by favorable enthalpic changes, as opposed to the isomerization step, which is solely contributed by entropic changes. The standard binding enthalpy (ΔH°) of SAHA, deduced from the transient kinetic as well as the isothermal titration calorimetric experiments, was 2-3 kcal/mol higher than that of TSA. The experimental data presented herein suggest that SAHA serves as a preferential (target-specific and -selective) HDAC8 inhibitor as compared to TSA. Arguments that the detailed kinetic and thermodynamic studies may guide the rational design of HDAC inhibitors as therapeutic agents are presented.

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2.

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg(2+) ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2-NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH (τ=0.4 ns) and bound NADH (τ=6.0 ns). We used this technique to investigate the effects of Mg(2+) on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the K(D) for NADH with ALDH2 ranged from 468 µM to 12 µM for Mg(2+) ion concentrations of 20 to 6000 µM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4s(-1) (6000 µM Mg(2+)) to 8.3s(-1) (0 µM Mg(2+)) as determined by addition of excess NAD(+) to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. The apparent NADH association/re-association rate constants were approximately 0.04 µM(-1)s(-1) over the entire Mg(2+) ion concentration range and demonstrate that Mg(2+) ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior of the enzyme activity as a function of Mg(2+) concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2-NADH complexes corresponding to free NADH, enzyme-bound NADH, and, potentially, an abortive NADH-enzyme-propanal complex (τ=11.2 ns).