Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 2....

Since hydride transfer is completely rate limiting for yeast formate dehydrogenase [Blanchard, J.S., & Cleland, W. W. (1980) Biochemistry 19, 3543], the intrinsic isotope effects on this reaction are fully expressed. Primary deuterium, 13C, and 18O isotope effects in formate and the alpha-secondary deuterium isotope effect at C-4 of the nucleotide have been measured for nucleotide substrates with redox potentials varying from -0.320 (NAD) to -0.258 V (acetylpyridine-NAD). As the redox potential gets more positive, the primary deuterium isotope effect increases from 2.2 to 3.1, the primary 13C isotope effect decreases from 1.042 to 1.036, the alpha-secondary deuterium isotope effect drops from 1.23 to 1.06, and Vmax decreases. The 18O isotope effects increase from 1.005 to 1.008 per single 18O substitution in formate (these values are dominated by the normal isotope effect on the dehydration of formate during binding; pyridinealdehyde-NAD gives an inverse value, possibly because it is not fully dehydrated during binding). These isotope effects suggest a progression toward earlier transition states as the redox potential of the nucleotide becomes more positive, with NAD having a late and acetyl-pyridine-NAD a nearly symmetrical transition state. By contrast, the I2 oxidation of formate in dimethyl sulfoxide has a very early transition state (13k = 1.0154; Dk = 2.2; 18k = 0.9938), which becomes later as the proportion of water in the solvent increases (13k = 1.0265 in 40% dimethyl sulfoxide and 1.0362 in water). alpha-secondary deuterium isotope effects with formate dehydrogenase are decreased halfway to the equilibrium isotope effect when deuterated formate is the substrate, showing that the bending motion of the secondary hydrogen is coupled to hydride transfer in the transition state and that tunneling of the two hydrogens is involved. The 15N isotope effect of 1.07 for NAD labeled at N-1 of the nicotinamide ring suggests that N-1 becomes pyramidal during the reaction. 18O fractionation factors for formate ion relative to aqueous solution are 1.0016 in sodium formate crystal, 1.0042 bound to Dowex-1, and 1.0040 as an ion pair (probably hydrated) in CHCl3. The CO2 analogue azide binds about 10(4) times better than the formate analogue nitrate to enzyme-nucleotide complexes (even though the Ki values for both and the affinity for formate vary by 2 orders of magnitude among the various nucleotides), but the ratio is not sensitive to the redox potential of the nucleotide. Thus, not the nature of the transition state but rather the shape of the initial binding pocket for formate is determining the relative affinity.(ABSTRACT TRUNCATED AT 400 WORDS)