Mechanism-based inactivation of serine transhydroxymethylases by D-fluoroalanine and related amino acids.

摘要: Serine transhydroxymethylase, from lamb or rabbit liver, is known to catalyze slow transamination of D-alanine, but not L-amino acids, in a tetrahydrofolate-independent reaction. Both enzymes will process the D-isomer beta-fluoroalanine for alpha, beta-elimination HF yield an aminoacrylate-pyridoxal-P-enzyme intermediate. This intermediate partitions between harmless hydrolysis pyruvate, NH4+, and active enzyme-pyridoxal-P (catalytic turnover) suicidal enzyme alkylation by covalent modification with average partition ratio 40-60 turnovers/inactivation event/monomer unit this tetrameric enzyme. Enzyme inactivation occurs stoichiometric incorporation radioactive label D-[1,2-14C]fluoroalanine. Titration enzymic cysteinyl --SH groups 5,5'-dithiobis(2-nitrobenzoate) indicates loss 1 group on inactivation. Acid radioactive-inactive confirms cysteine residue modification. Treatment inactive 6 M urea, then KBH4, followed acid yields two compounds, lanthionine S-carboxyhydroxyethylcysteine, about equal amounts. The addition tetrahydrofolate stimulates both pyruvate production extents 200-fold rate acceleration at 0.5 mM turnover numbers approximately 120 min-1. Km D-fluoroalanine high, 10-60 mM, low substrate affinity suggests be useful vivo agent selective liver cell serine transhydroxymethylases.