(1-Aminoethyl)boronic acid: a novel inhibitor for Bacillus stearothermophilus alanine racemase and Salmonella typhimurium D-alanine:D-alanine ligase (ADP-forming).
(1-Aminoethyl)boronic acid (Ala-B), an analogue of alanine in which a boronic acid group replaces the carboxyl group, has been synthesized and found to inhibit the first two enzymes, alanine racemase (from Bacillus stearothermophilus, EC 5.1.1.1) and D-alanine:D-alanine ligase (ADP-forming) (from Salmonella typhimurium, EC 6.3.2.4), of the D-alanine branch of bacterial peptidoglycan biosynthesis. In both cases, time-dependent, slow binding inhibition is observed due to the generation of long-lived, slowly dissociating complexes. Ala-B inhibits alanine racemase with a Ki of 20 mM and a kappa inact of 0.15-0.35 min-1. Time-dependent loss of activity is paralleled by conversion of the 420-nm chromophore of initial bound PLP aldimine to a 324-nm absorbing species. On dilution of Ala-B, racemase activity is regained with a t1/2 of ca. 1 h. The D-Ala-D-Ala ligase also shows progressive inhibition by Ala-B provided ATP (but not AMP-PNP or AMP-PCP) is present. The presence of D-alanine along with ATP also leads to Ala-B-induced inactivation. Kinetic analysis suggests Ala-B can compete with D-alanine at either of the two D-alanine binding sites, and on inactivation with Ala-B, labeled D-alanine, and labeled ATP, the inactive enzyme has stoichiometric amounts of D-alanine, ADP, Pi, and Ala-B bound. The half-life of inactive enzyme complexes varied from approximately 2 h (without D-alanine) to 4.5 days (with D-alanine). No D-Ala-D-Ala-B dipeptide was detected.(ABSTRACT TRUNCATED AT 250 WORDS)
Duncan
K
K
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Faraci
W S
WS
Matteson
D S
DS
Walsh
C T
CT
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000411:Alanine Racemase / Q000037:antagonists & inhibitors*; D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D001896:Boron Compounds / Q000494:pharmacology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*; D012486:Salmonella typhimurium / Q000201:enzymology
D001896:Boron Compounds; C060287:(1-aminoethyl)boronic acid; D000255:Adenosine Triphosphate; D000591:Amino Acid Isomerases; D000411:Alanine Racemase; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase