Probing the Role of the Conserved Arg174 in Formate Dehydrogenase by Chemical Modification and Site-Directed Mutagenesis.

The reactive adenosine derivative, adenosine 5'--[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD. AMPS-HDB was used as a chemical label towards formate dehydrogenase (FDH). AMPS-HDB reacts covalently with FDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of FDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (K = 0.66 ± 0.15 mM, first order maximum rate constant k = 0.198 ± 0.06 min). NAD and NADH protects FDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme FDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The FDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of FDH.