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

ABSTRACT: The reactive adenosine derivative, adenosine 5'-O-[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 Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (K_D = 0.66 ± 0.15 mM, first order maximum rate constant k₃ = 0.198 ± 0.06 min^-1). NAD⁺ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the re

SUBMITTER: Alqarni MH 

PROVIDER: S-EPMC7956174 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature