Malate dehydrogenase, anticooperative NADH, and L-malate binding in ternary complexes with supernatant pig heart enzyme

Abstract

Supernatant malate dehydrogenase from pig heart, a dimeric protein containing two very similar or identical subunits, shows negatively cooperative (anticooperative) interactions between NADH binding sites in the presence, but not in the absence, of 0.1 M L-malate. This behavior is observed consitently whether the technique used employs protein fluorescence quenching, NADH fluorescence enhancement, or ultrafiltration dialysis. Fluorescence titration shows that L-malate is also anticooperatively bound in the presence of saturating concentrations of NADH. The data are consistent with an "induced asymmetry" model in which conformational change accompanies the formation of the ternary complex. Two of the three chromatographically resolvable forms of the enzyme have been tested and found to have anticooperative behavior.

Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide–adenine dinucleotide-binding studies

“…This is but one example of such results obtained with both pig and ox enzyme, at pH 6 and pH 8 and over a wide range of protein concentrations. It confirms the results of Cassman &King (1972) andof Mueggler et al (1975). Linear changes in protein fluorescence when NADH binds to the oligomeric dehydrogenase are unusual (Holbrook, 1972) and require explanation.…”

“…and s-MDHb); the forms of the enzyme have identical amino acid composition and molecular weights. Mueggler et al (1975) report that negative co-operative interactions between the NADH-binding sites of the pig heart soluble enzyme T are induced by L-malate, and that at saturating concentrations of NADH L-malate binds with negative co-operativity.…”

“…The binding of NADH to pig and ox heart malate dehydrogenase was monitored by the decreased protein fluorescence at pH 8.0 in 0.1 M-L-malate as described by Mueggler et al (1975). However, NADH binds only weakly at pH8.0 (Holbrook & Wolfe, 1972), and the decrease in protein fluorescence caused by added NADH must be resolved into two components, (a) the 'trivial' decrease caused by absorption of the incident (297nm) and fluorescent (340nm) light by the added soluble NADH ('inner filter effect') and (b) the decrease caused by NADH bound to the protein.…”

Mechanism of complexing of chlorin e 6 and tetra(p-carboxyphenyl)porphyrin with malate dehydrogenase

“…However, according to their data, steric barriers to the bonding of the coenzyme to the second MDG subunit arise only in the presence of an enzyme substrate (L-malate molecules) in the solution. In this case, the Hill coefficient h = 0.64 (negative cooperativity), while in the absence of L-malate the bonding of NADH is, according to the data of [32], noncooperative (h = 0.97). Note that the bonding of other specific ligands of the enzyme, such as its substrates or inhibitors, is, according to [31], noncooperative.…”

“…According to [30,31], the complexing of MDG with the coenzymes NADH and NAD + is characterized by negative cooperativity. P. A. Mueggler et al [32] also pointed to the existence of significant interactions between the sites of bonding of NADH on the MDG molecule. However, according to their data, steric barriers to the bonding of the coenzyme to the second MDG subunit arise only in the presence of an enzyme substrate (L-malate molecules) in the solution.…”