Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study.
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.
De Weck
Z
Z
Biochemisches Institut, Universität Zürich, Switzerland.
Pande
J
J
Kägi
J H
JH
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D003903:Deuterium; D006859:Hydrogen; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008024:Ligands; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013055:Spectrophotometry, Infrared; D013552:Swine
D008024:Ligands; D009243:NAD; D006859:Hydrogen; D003903:Deuterium; D000426:Alcohol Dehydrogenase; D007770:L-Lactate Dehydrogenase