Enzymatic and thermodynamic profiles of a heterotetramer lactate dehydrogenase isozyme in swine

Abstract

Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD⁺. In somatic cells, LDH forms homotetramers and heterotetramers that are encoded by two different genes: LDHA (skeletal muscle type, M) and LDHB (heart type, H). Analysis of LDH isozymes is important for understanding the physiological role of homotetramers and heterotetramers and for optimizing inhibition of their enzymatic activity as it may result in distinct effects. Previously, we reported that hydroxychloroquine (HCQ) inhibited LDH activity, but we did not examine isozyme specificity. In the present study, we isolated heterotetrameric LDH (H₂M₂) from swine brain, determined its kinetic and thermodynamic properties, and examined the effect of HCQ on its activity compared to homotetrameric LDH isozymes. We show that: (1) the K_m values for H₂M₂-mediated catalysis of pyruvate or lactate were intermediate compared to those for the homotetrameric isozymes, M₄ and H₄ whereas the V_max values were similar; (2) the K_m and V_max values for H₂M₂-mediated catalysis of NADH were not significantly different among LDH isozymes; (3) the values for activation energy and van't Hoff enthalpy changes for pyruvate reduction of H₂M₂ were intermediate compared to those for the homotetrameric isozymes; (4) the temperature for half residual activity of H₂M₂ was closer to that for M₄ than for H₄. We also show that HCQ had different affinities for various LDH isozymes.

Keywords: H(2)M(2); HCQ; Heterotetrameric isozyme; Hydroxychloroquine; K(m); LDH; Lactate dehydrogenase; T(half); T(max); V(max); heterotetrameric lactate dehydrogenase isozyme consisting of two LDHA-encoded muscle type and two LDHB-encoded heart muscle type molecules; hydroxychloroquine; lactate dehydrogenase; optimum temperature for LDH pyruvate reduction activity; residual activity of LDH pyruvate reduction activity; van't Hoff enthalpy.