Characterization of the NAD(+) binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis

The 2',3'-dialdehyde derivative of ADP (oADP) has been shown to be an affin ity label for the NAD(+) binding site of recombinant Candida boidinii forma te dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biph asic pseudo first-order saturation kinetics. The rate of inactivation exhib ited a nonlinear dependence on the concentration of oADP, which can be desc ribed by reversible binding of reagent to the enzyme (K-d = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction , with maximum rate constants of 0.012 and 0.007 min(-1) for the fast and s low phases, respectively. Inactivation of formate dehydrogenase by oADP res ulted in the formation of an enzyme-oADP product, a process that was revers ed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactiva tion). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme-oADP complex was previously reduced by NaBH4, suggesting that t he reaction product was a stable Schiff's base. Protection from inactivatio n was afforded by nucleotides (NAD(+), NADH and ADP) demonstrating the spec ificity of the reaction. When the enzyme was completely inactivated, approx imately 1 mol of [C-14]oADP per mol of subunit was incorporated. Cleavage o f [C-14]oADP-modified enzyme with trypsin and subsequent separation of pept ides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of th e radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific rea ction at the nucleotide binding site, with negative cooperativity between s ubunits accounting for the appearance of two phases of inactivation. Molecu lar modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 pro gram. The model confirmed that Lys360 is positioned at the NAD(+)-binding s ite. Site-directed mutagenesis was used in dissecting the structure and fun ctional role of Lys360. The mutant Lys360 --> Ala enzyme exhibited unchange d k(cat) and K-m values for formate but showed reduced affinity for NAD(+). The molecular model was used to help interpret these biochemical data conc erning the Lys360 --> Ala enzyme. The data are discussed in terms of engine ering coenzyme specificity.