Time-dependent inhibition of Bacillus stearothermophilus alanine racemase by (1-aminoethyl)phosphonate isomers by isomerization to noncovalent slowly dissociating enzyme-(1-aminoethyl)phosphonate complexes.
An alanine racemase encoded by a gene from the thermophilic Gram-positive bacterium Bacillus stearothermophilus is overproduced to 0.3% of the soluble protein when carried on plasmid pICR4 in Escherichia coli [Inagaki, K., Tanizawa, K., Badet, B., Walsh, C. T., Tanaka, H., & Soda, K. (1986) Biochemistry (third paper of four in this issue)]. Purification of large quantities (50 mg) of racemase permits study of time-dependent inactivation by D and L isomers of the antibacterial (1-aminoethyl)phosphonate (Ala-P), the phosphonate analogue of alanine. The time-dependent activity loss by this compound now appears general to Gram-positive but not to Gram-negative racemases [Badet, B., & Walsh, C. (1985) Biochemistry 24, 1333] and is shown to occur by extremely slow dissociation of a noncovalent E X Ala-P complex. Ala-P binds initially in a weak, reversible (KI = 1 mM) competitive manner but is slowly isomerized (kinact = 6-9 min-1) to a stoichiometric enzyme complex, which in turn dissociates extremely slowly, with a half-time about 25 days. Thus, Ala-P is a slow but not a tight-binding inhibitor. The E X Ala-P complex is not reducible by borohydride but does perturb the fluorescence of bound pyridoxal 5'-phosphate coenzyme. Determination of the sequence of an active site octapeptide of the B. stearothermophilus alanine racemase shows homology with the sequence of a Gram-negative Salmonella typhimurium alanine racemase that is not susceptible to time-dependent inhibition by Ala-P. Studies with Ala-P analogues suggest the phosphonate dianion is crucial for stable formation of an isomerized long-lived E X Ala-P-inhibited complex.
D000411:Alanine Racemase / Q000302:isolation & purification*; D000591:Amino Acid Isomerases / Q000302:isolation & purification*; D000615:Aminoethylphosphonic Acid / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007536:Isomerism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D011485:Protein Binding
D009943:Organophosphorus Compounds; D000615:Aminoethylphosphonic Acid; D000591:Amino Acid Isomerases; D000411:Alanine Racemase