Mechanism-based inactivation of alanine racemase by 3-halovinylglycines.
Alanine racemase, an enzyme important to bacterial cell wall synthesis, is irreversibly inactivated by 3-chloro- and 3-fluorovinylglycine. Using alanine racemase purified to homogeneity from Escherichia coli B, the efficient inactivation produced a lethal event for every 2.2 +/- 0.2 nonlethal turnovers, compared to 1 in 800 for fluoroalanine. The mechanism of inhibition involves enzyme-catalyzed halide elimination to form an allenic intermediate that partitions between reversible and irreversible covalent adducts, in the ratio 3:7. The reversible adduct (lambda max = 516 nm) decays to regenerate free enzyme with a half-life of 23 min. The lethal event involves irreversible alkylation of a tyrosine residue in the sequence -Val-Gly-Tyr-Gly-Gly-Arg. The second-order rate constant for this process with D-chlorovinylglycine (122 +/- 14 M-1 s-1), the most reactive analog examined, is faster than the equivalent rate constant for D-fluoroalanine (93 M-1 s-1). The high killing efficiency and fast turnover of these mechanism-based inhibitors suggest that their design, employing the haloethylene moiety to generate a reactive allene during catalysis, could be extended to provide useful inhibitors of a variety of enzymes that conduct carbanion chemistry.
Department of Enzymology, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065.
N A
NA
Thornberry
H G
HG
Bull
D
D
Taub
K E
KE
Wilson
G
G
Giménez-Gallego
A
A
Rosegay
D D
DD
Soderman
A A
AA
Patchett
D000411:Alanine Racemase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D013053:Spectrophotometry; D013237:Stereoisomerism
C055749:fluorovinylglycine; C055748:chlorovinylglycine; D000411:Alanine Racemase; D005998:Glycine