N-tosyl-L-phenylalanine chloromethyl ketone, a serine protease inhibitor, identifies glutamate 398 at the coenzyme-binding site of human aldehyde dehydrogenase. Evidence for a second "naked anion" at the active site.
Human aldehyde dehydrogenase isozymes were inactivated by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of chymotrypsin. The inactivation was a first-order process that followed saturation kinetics. NAD and chloral when used together protected against inactivation. In steady-state kinetics, TPCK produced only slope effects versus varied NAD, both slope and intercept effects versus varied glycolaldehyde were produced, indicating that TPCK reacted with the same enzyme form with which NAD reacted. Ki values from steady-state kinetics and saturation kinetics were comparable. Use of [3H]-labeled TPCK showed that inactivation was associated with the incorporation of two molecules of TPCK per molecule of enzyme. The label incorporation occurred into a single tryptic peptide and also into a single chymotryptic peptide of the E1 isozyme. Purification of labeled peptides, followed by sequencing, demonstrated that E398 of aldehyde dehydrogenase was labeled. Reaction of a haloketone, TPCK, with a carboxyl group of E398 indicates that E398 occurs as a "naked anion" within the molecule. This paper constitutes identification of the second (after E268) "naked anion" at the active site of aldehyde dehydrogenase.
Dryjanski
M
M
Center of Alcohol Studies, Rutgers The State University of New Jersey, Piscataway 08854-8001, USA.
Kosley
L L
LL
Pietruszko
R
R
D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin; D003593:Cytoplasm / Q000201:enzymology; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin
D007527:Isoenzymes; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D009243:NAD; D018698:Glutamic Acid; D014108:Tosylphenylalanyl Chloromethyl Ketone; D000444:Aldehyde Dehydrogenase; D002918:Chymotrypsin; D014357:Trypsin