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

Abstract

The 2 H ,3 H -dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD 1 binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described 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 21 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme±oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (. 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme±oADP complex was previously reduced by NaBH 4 , suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD 1 , NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [ 14 C]oADP per mol of subunit was incorporated. Cleavage of [ 14 C]oADPmodified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular 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 program. The model confirmed that Lys360 is positioned at the NAD 1 -binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys3603Ala enzyme exhibited unchanged k cat and K m values for formate but showed reduced affinity for NAD 1 . The molecular model was used to help interpret these biochemical data concerning the Lys3603Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.

Catalytic mechanism and application of formate dehydrogenase

“…Shortcomings of FDHs are their rather low specific activities: 6 7 and 10 units/mg protein for homogenous FDHs from yeasts [18,95] and bacteria [71], respectively (Table 1). Moreover, NADP + specific FDHs have not yet been found in nature, and this prevents using this enzyme for regeneration of NADPH.…”

S‐(2,3‐Dichlorotriazinyl)glutathione

“…For both phases, a plot of 1/ k obs vs. 1/[SDTG] yields a straight line. This indicates that the reaction obeys pseudo‐first‐order saturation kinetics and is consistent with reversible binding of reagent before covalent modification according to the following equation [17–20]: where E represents the free enzyme, E:SDTG is the reversible complex, and E‐SDTG is the covalent product. The steady‐state rate equation for the interaction is [23–25]: where k obs is the rate of enzyme inactivation for a given concentration of SDTG, k 3 is the maximal rate of inactivation (min −1 ), and K d is the apparent dissociation constant of the E:SDTG complex.…”

“…GST I was inactivated at 25 °C in 1 mL incubation mixture containing potassium phosphate buffer, pH 6.5 (100 µmol), SDTG (0–218.2 nmol) and enzyme (2 units, GST assay at 30 °C). The rate of inactivation was followed by periodically removing samples (20 µL) for assay of enzymatic activity [17,20].…”

“…Affinity labelling is a useful tool for the identification and probing of specific catalytic and regulatory sites in purified enzymes and proteins [17–20]. Affinity labelling experiments complement the results from crystallography and provide structural information on proteins in free solution.…”

The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

“…The rate of inactivation was followed by periodically removing samples (5–20 µL) for assay of enzymatic activity [30], [31]. Rate constants for the reaction exhibiting biphasic kinetics were calculated using the equation [32], [33]:where F represents the fractional residual activity of the partial active enzyme intermediate.…”