Identification of the active-site serine in human lecithin: cholesterol acyltransferase.
Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003488:Cyanogen Bromide / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D009943:Organophosphorus Compounds; D010455:Peptides / Q000032:analysis; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000032:analysis* / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D012694:Serine / Q000032:analysis*; D013997:Time Factors
D000596:Amino Acids; D009943:Organophosphorus Compounds; D010455:Peptides; C002226:diisopropylphosphate; D012694:Serine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D003488:Cyanogen Bromide