Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride

Palmitoyl-protein thioesterase-1 (PPT1) is a newly de scribed lysosomal enz yme that hydrolyzes long chain fatty acids from lipid-modified cysteine res idues in proteins, Deficiency in this enzyme results in a severe neurodegen erative storage disorder, infantile neuronal ceroid lipofuscinosis. Althoug h the primary structure of PPT1 contains a serine lipase consensus sequence , the enzyme is insensitive to commonly used serine-modifying reagents phen ylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the cur rent paper, we show that the active site serine in PPT1 is modified by a su bstrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K-t of the inhibition was 125 mu M (in t he presence of 1.5 mM Triton X-100), and the catalytic rate constant for su lfonylation (k(2)) was 3.3/min, a value similar to previously described sul fonylation reactions. PPT1 was crystallized after inactivation with HDSF, a nd the structure of the inactive form was determined to 2.4 Angstrom resolu tion. The hexadecylsulfonyl was found to modify serine 115 and to snake thr ough a narrow hydrophobic channel that would not accommodate an aromatic su lfonyl fluoride, Therefore, the geometry of the active site accounts for th e reactivity of PPT1 with HDSF but not PMSF, These observations suggest a s tructural explanation as to why certain serine lipases are resistant to mod ification by commonly used serine-modifying reagents.