Tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) binds successively four molecules of its cofactor (NAD + ) with affinities of ca 10 11 M −1 , 10 9 M −1 , 10 7 M −1 , and 10 5 M −1 . The reduction in the dynamics of the protein is greatest upon binding the first NAD + molecule. Smaller reductions then occur upon binding the second and third NAD + molecules, and the fourth NAD + molecule binds without dynamic change. Reduction of the GAPDH dynamics, with consequent improvements in its internal bonding, can account for the increase in NAD + binding affinity from 10 5 M −1 to 10 11 M −1 . Evidence is provided that comparable fractions of the binding energy of other ligands, and of the catalytic efficiency of enzymes, may be derived in the same way.