Premium
Identification Of Cysteine Residues In ALDH2 Responsible For Its Bioactivation Of Nitroglycerin
Author(s) -
Tsou PeiSuen,
Page Nathaniel A.,
Qu Jun,
Fung HoLeung
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb375
Subject(s) - aldh2 , chemistry , aldehyde dehydrogenase , biochemistry , esterase , dehydrogenase , cysteine , enzyme , trypsin , alcohol dehydrogenase
The dehydrogenase, esterase, and nitrate‐bioactivating activities of mitochondrial aldehyde dehydrogenase ALDH2 were significantly inactivated by nitroglycerin (NTG). Tandem mass spectrometry sequencing of a trypsin or glu‐c‐digested NTG+ALDH2 mixture, after reduction by tris(2‐carboxyethyl)phosphine, indicated that oxidation occurred at C66 (to ‐SOH) and C386 (to ‐SOH, ‐SO 2 H, ‐SO 3 H). Modification at C319 was not detected, probably due to the low ionization efficiency of the C319‐contained tryptic or Glu‐c domains. Mutants of C66A, C386A, and C319A (the active site for dehydrogenase and esterase activities) were constructed. The specific ALDH activities (SA) of the WT, C66A, C386A and C319A were 2.51, 2.25, 3.56, 0 U/mg protein, respectively. Within 2 min of incubation, NTG was completely converted into 1,2‐glyceryl dinitrate (GDN) by WT, C66A, and C386A, but not by C319A.. None of these enzymes produced 1,3‐GDN. Consistent with these results, ALDH2 WT, C66A, and C386A catalyzed NO production from NTG with similar activities (cumulative NO production in μM*sec/SA: WT: 60.1 ± 15, C66A: 57.5 ± 24, C386A: 72.2 ± 32), while C319A did not liberate any detectable NO. These results suggest that the dehydrogenase, esterase, and NTG‐bioactivating activities of ALDH2 share the same active C319 site. (Supported in part by NIH HL81580).