Thermodynamic and kinetic experiments have been performed at ionic strength 0.30 to elucidate the relationship between the structure of pig heart H₄-LDH (lactate dehydrogenase) and its catalytic function. Calorimetry and fluorescence were used to determine all the thermodynamic parameters for binary and ternary complex formation. TFL (trifluorolactate) and oxamate were employed as nonreactive analogs of the substrates lactate and pyruvate, respectively, to examine ternary complex formation in the absence of the ensuing redox step. At pH 6 where there is no apparent change in the protonation state of LDH upon binary complex formation, LDH binds NADH more tightly than NAD due to an entropy effect, i.e., only 1.1 out of the 3.1 kcal/mole difference in free energy changes is enthalpic in origin. The heat capacities of LDH·NAD (-150 ± 30 cal/K-mole) and LDH·NADH (-220 ± 40 cal/K-mole) formation at pH 6 and 25°C are relatively small and similar. These results suggest the importance of charge interactions in coenzyme binding. Structural information indicates that Arg-106, a positively charged residue of a loop of polypeptide in LDH which at equilibrium alternates between two conformations, open (extended into solvent) and closed (part of the active site), interacts unfavorably with the positively charged nicotinamide ring of NAD when the loop is in the closed conformation. Thermodynamic experiments demonstrate the suitability of TFL as an analog of lactate. TFL displays the correct specificity by binding to LDH·NAD more tightly (Kₐ = 400 M⁻¹) than to LDH·NADH (Kₐ = 34 M⁻¹) at pH 8 and 25°C. This binding requires that an enzymic residue with a pK = 6.7 not be protonated in accordance with the role of His-193 in analog binding in crystalline ternary complexes. Since the free energy change of the redox step is small, the difference in the free energy changes of formation of LDH·NAD·TFL and LDH·NADH·oxamate from LDH+NAD+TFL and LDH+NADH+oxamate, respectively, should approximate the free energy change of the actual enzymic reaction. The free energy and enthalpy changes of this model reaction are within 10% of the values of the actual reaction. Steady-state kinetic experiments further support the use of TFL as an analog of lactate. At pH 8 and 25°C TFL acts mainly as competitive inhibitor of lactate during lactate oxidation. The difference between the TFL dissociation constant (2.5 mM) and its inhibition constant (8.0 mM) means that TFL is not a simple dead-end inhibitor, i.e., LDH·NAD·TFL must be connected to the productive pathway of the reaction at more than one point. This is consistent with the existence of two conformational states of LDH·NAD. Additional support for the existence of two conformational states of LDH comes from analysis of the heat capacity changes of ternary complex formation. The large negative heat capacity changes at 25°C of TFL binding to LDH·NAD at pH 8 (-150 cal/K-mole) and of oxamate binding to LDH·NADH at pH 8 (-330 cal/K-mole) and pH 6 (-420 cal/K-mole) are partly attributed to a reaction heat effect arising from a shift in the conformational equilibrium of LDH to one in which the loop is in the closed position. As shown by calorimetry and fluorescence, phosphate binds to a single class of sites of LDH. The thermodynamic parameters of this process at pH 6 and 25°C are ΔG = -30 kcal/mole, ΔH = -5.1 kcal/mole, and ΔS = -7.0 cal/K-mole. Binding is not at the active site.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/290527 |
| Date | January 1980 |
| Creators | O'Neal, Clifford Cecil |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
Page generated in 0.0016 seconds