Steady-state kinetic characterization and mechanistic modeling of human lactate dehydrogenase A as a reference framework for inhibitor screening

Human lactate dehydrogenase A (hLDH-A) is a key enzyme in cancer-associated metabolic reprogramming, catalyzing the reversible interconversion of pyruvate and lactate coupled with the NADH/NAD+ redox pair. A rigorous kinetic reference is therefore essential for the reliable evaluation of hLDH-A modulation, particularly in the context of enzyme-based drug screening. In this study, the steady-state kinetics of hLDH-A were systematically characterized under physiological-like conditions by analyzing both forward and reverse reactions. Apparent kinetic parameters were determined using the Michaelis–Menten model and Hanes–Woolf linearization, complemented by nonlinear fitting of untransformed data, while intrinsic kinetic constants were extracted assuming an ordered sequential bi–bi mechanism. The results revealed a pronounced preference for the pyruvate-to-lactate reaction, driven primarily by cofactor-dependent steps rather than substrate affinity. The established kinetic framework was subsequently applied to the comparative evaluation of reference LDH inhibitors, enabling discrimination among competitive, non-competitive, and mixed inhibition mechanisms and the determination of inhibition constants. In particular, oxamate exhibited competitive inhibition relative to pyruvate, whereas galloflavin, gossypol, and FX11 exhibited predominantly non-competitive behavior. NHI-2 showed a mixed inhibition pattern. Inhibition constants, determined under pyruvate-variation conditions, ranged from 6.9 to 52.5 μM, with gossypol showing the highest inhibitory potency among the investigated compounds. Based on the determined kinetic parameters, a mechanistic model describing the net reaction rate was developed to simulate the temporal evolution of the enzymatic system. The model captured the main trend of NADH consumption observed experimentally and qualitatively reproduced the distinct kinetic effects associated with competitive and non-competitive inhibition mechanisms