Purification of an alanine racemase from Streptococcus faecalis and analysis of its inactivation by (1-aminoethyl)phosphonic acid enantiomers.
An alanine racemase has been purified some 30 000-fold almost to homogeneity from Gram-positive Streptococcus faecalis NCIB 6459; the enzyme has been purified to the same extent (4000-fold) from an O-carbamyl-D-serine-resistant mutant with a 7-fold higher enzyme level in crude extract. The racemase has one pyridoxal phosphate molecule per 42-kDa subunit, has a Vmax of 3570 units/mg and a Km of 7.8 mM in the L to D direction, and has a Vmax of 1210 units/mg and a Km of 2.2 mM in the D to L direction. The Keq is 0.8 and kcat/Km values are ca. 3 X 10(5) M-1 s-1. The purified enzyme is inhibited in a time-dependent manner by both L- and D-(l-aminoethyl)phosphonates (Ala-P), confirming observations of Atherton et al. in crude extracts of this organism [Atherton, F. R., Hall, M. J., Hassal, C. H., Holmes, S. W., Lambert, R. W., Lloyd, W. J., & Ringrose, P. S. (1980) Antimicrob. Agents Chemother. 18, 897]. Studies with [1-2H]-, [1-3H]-, and [1,2-14C]Ala-P rule out enzymic activation and processing as the basis for irreversible inhibition. Thus, enzyme after exposure to [14C]Ala-P or [alpha-3H]Ala-P and gel filtration contains stoichiometric amounts of radioactive label, but denaturation quantitatively releases intact Ala-P into solution as revealed by high-performance liquid chromatography and cocrystallization with authentic material. The Ala-P isomers are slow binding inhibitors of this racemase as is the alpha,alpha'-dimethyl analogue but not the D or L isomers of the corresponding phosphinate.(ABSTRACT TRUNCATED AT 250 WORDS)
D000411:Alanine Racemase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000591:Amino Acid Isomerases / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000615:Aminoethylphosphonic Acid / Q000494:pharmacology*; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000201:enzymology*; D007536:Isomerism; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000494:pharmacology*
D000596:Amino Acids; D009943:Organophosphorus Compounds; D000615:Aminoethylphosphonic Acid; D000591:Amino Acid Isomerases; D000411:Alanine Racemase