PARACATALYTIC INACTIVATION OF L-2-HALOACID DEHALOGENASE FROM PSEUDOMONAS SP. YL BY HYDROXYLAMINE - EVIDENCE FOR THE FORMATION OF AN ESTER INTERMEDIATE

Asp(10) of L-2-haloacid dehalogenase from Pseudomonas sp. YL was propo sed to act as a nucleophile to attack the alpha-carbon of L-2-haloalka noic acids to form an ester intermediate, which is hydrolyzed by nucle ophilic attack of a water molecule on the carbonyl carbon (Liu, J.-Q, Kurihara, T., Miyagi, M., Esaki, N., and Soda, K. (1995) J. Biol. Chem . 270, 18309-18312). We have found that the enzyme is paracatalyticall y inactivated by hydroxylamine in the presence of the substrates monoc hloroacetate and L-2-chloropropionate. Ion spray mass spectrometry dem onstrated that the molecular mass of the enzyme inactivated by hydroxy lamine during the dechlorination of monochloroacetate is about 74 Da g reater than that of the native enzyme. To determine the increase of th e molecular mass more precisely, we digested the inactivated enzyme wi th lysyl endopeptidase and measured the molecular masses of the peptid e fragments. The molecular mass of the hexapeptide Gly(6)-Lys(11) was shown to increase by 73 Da. Tandem mass spectrometric analysis of this peptide revealed that the increase is due to a modification of Asp(10 ). When the enzyme was paracatalytically inactivated by hydroxylamine during the dechlorination of L-2-chloropropionate, the molecular mass of the hexapeptide was 87 Da higher. Hydroxylamine is proposed to atta ck the carbonyl carbon of the ester intermediate and form a stable asp artate beta-hydroxamate carboxyalkyl ester residue in the inactivated enzyme.