PMID- 10801859 OWN - NLM STAT- MEDLINE DCOM- 20000907 LR - 20210209 IS - 0021-9258 (Print) IS - 0021-9258 (Linking) VI - 275 IP - 31 DP - 2000 Aug 4 TI - Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride. PG - 23847-51 AB - Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents. FAU - Das, A K AU - Das AK AD - Department of Internal Medicine and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593, USA. FAU - Bellizzi, J J 3rd AU - Bellizzi JJ 3rd FAU - Tandel, S AU - Tandel S FAU - Biehl, E AU - Biehl E FAU - Clardy, J AU - Clardy J FAU - Hofmann, S L AU - Hofmann SL LA - eng SI - PDB/1EXW GR - CA59021/CA/NCI NIH HHS/United States GR - NS35323/NS/NINDS NIH HHS/United States GR - RR-01646/RR/NCRR NIH HHS/United States PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - J Biol Chem JT - The Journal of biological chemistry JID - 2985121R RN - 0 (Alkylating Agents) RN - 0 (Recombinant Proteins) RN - 0 (Sulfones) RN - 57KD15003I (Phenylmethylsulfonyl Fluoride) RN - 86855-26-7 (hexadecanesulfonyl fluoride) RN - EC 3.1.2.- (Thiolester Hydrolases) RN - EC 3.1.2.2 (Palmitoyl-CoA Hydrolase) RN - EC 3.1.2.22 (palmitoyl-protein thioesterase) SB - IM MH - Acylation MH - Alkylating Agents/pharmacology MH - Animals MH - Catalytic Domain MH - Cattle MH - Lysosomes/enzymology MH - Models, Molecular MH - Molecular Sequence Data MH - Neuronal Ceroid-Lipofuscinoses/etiology MH - Palmitoyl-CoA Hydrolase/drug effects MH - Phenylmethylsulfonyl Fluoride/*pharmacology MH - Recombinant Proteins/drug effects MH - Sulfones/pharmacology MH - Thiolester Hydrolases/*drug effects/genetics EDAT- 2000/05/10 09:00 MHDA- 2000/09/09 11:01 CRDT- 2000/05/10 09:00 PHST- 2000/05/10 09:00 [pubmed] PHST- 2000/09/09 11:01 [medline] PHST- 2000/05/10 09:00 [entrez] AID - S0021-9258(19)66050-3 [pii] AID - 10.1074/jbc.M002758200 [doi] PST - ppublish SO - J Biol Chem. 2000 Aug 4;275(31):23847-51. doi: 10.1074/jbc.M002758200.