Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study

Abstract

Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change of measured by exchange retardation is considerably larger for NAD+ than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD+-pyrazole, PHLDH-NAD+-oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD+ is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change.

Yeast Alcohol Dehydrogenase Structure and Catalysis

“…85 Hydrogen–deuterium exchange studies also suggest that binding of NAD + results in a conformational change. 86 …”

“…85 Hydrogen−deuterium exchange studies also suggest that binding of NAD + results in a conformational change. 86 The next step in the mechanism, the binding of alcohol to the enzyme−NAD + complex, may occur by direct displacement of Glu-67 by the alcohol or by a double displacement of a water that could bind to the zinc in the enzyme−NAD + complex. The homologous E. coli enzyme complexed with NAD has water bound to the catalytic zinc.…”

NGF inhibits human leukemia proliferation by downregulating cyclin A1 expression through promoting acinus/CtBP2 association

“…This observation might explain the unsuccessful interaction between full-length CtBP2 and acinus-S in vitro (Fig 1A), which could be rescued in the presence of NAD + (Fig 2A). Since conformational change upon NAD + binding is a key feature of NAD + -dependent dehydrogenase (De Weck et al , 1987), binding of NAD + to CtBP2 would somehow stabilize the C-terminal structure of CtBP2, which favors its binding to acinus-S.…”

Evidence for temperature-dependent conformational changes in the L-lactate dehydrogenase from Bacillus stearothermophilus

“…A comparison of the immobilization of aromatic residues (measured by near-UV CD) in the presence of FBP with that in the absence of FBP also points to a more flexible structure without FBP above 60 °C (data not shown). Numerous studies have shown that the accessibility of peptide hydrogens to solvent is influenced by ligand binding, suggesting that the ligands impose constraints on the dynamics of protein structures (De Week et al, 1987, and references cited therein). It is therefore not astonishing that the point of thermal denaturation of BSLDH is increased by 3 to 5 °C in the presence of FBP (data not shown).…”