Sulfonyl Fluorides as Inhibitors of Esterases. II. Formation and Reactions of Phenylmethanesulfonyl α-Chymotrypsin<sup>*</sup>

Gold, Allen M.; Fahrney, David E.

doi:10.1021/bi00894a009

Cited by 295 publications

(122 citation statements)

References 27 publications

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“…The presence of the serine consensus motif in the esterase confirmed that this enzyme is a typical serine hydrolase which performs hydrolysis of ester bonds after nucleophilic attack of the hydroxyl group from the active serine residue. Studies of inhibition by PMSF, a serinehydrolase inhibitor (Gold & Fahrney, 1964;Kraut, 1977), further support the involvement of the active serine in the catalytic mechanism of the esterase (unpublished results).…”

Section: Discussionmentioning

confidence: 78%

A bacterial esterase is homologous with nonhaem haloperoxidases and displays brominating activity

Pelletier

Altenbuchner

1995

Microbiology

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Screening GenBank indicated that an esterase from Pseudomonas fluorescens had high sequence similarity with bacterial non-haem haloperoxides. However, this homology was limited to two distinct domains of the published esterase sequence. As errors in the published sequence were suspected, the esterase gene was sequenced again. The revised sequence displayed between 40 and 50% identical amino acids with the haloperoxidases, but distributed along the whole sequence. In addition to the structural homologies with haloperoxidases, the esterase also displayed functional homology. The recombinant esterase, purified from Escherichia coli cells, was capable of both ester hydrolysis and halogenation, as detected in situ by the formation of bromophenol blue or spectrophotometrically by the bromination of monochlorodimedon. The esterase is thus a bifunctional enzyme. The sequence analysis and the biochemical investigations show that the esterase belongs to the haloperoxidase family. It also possessed, however, a typical feature of serine-hydrolases, namely the consensus motif Gly-X-Ser-X-Gly around the active serine of the catalytic triad. By alignment of the esterase with different serine-hydrolase sequences, it was possible to identify the other two residues of the triad. The triad comprised the residues Ser95, Asp223 and His252. Interestingly, a structurally equivalent catalytic triad was also identified in the sequences of all bacterial non-haem haloperoxidases, in highly conserved domains. The presence of a catalytic triad in haloperoxidases is expected to be important in the mechanism of halogenation.

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Section: Discussionmentioning

confidence: 78%

A bacterial esterase is homologous with nonhaem haloperoxidases and displays brominating activity

Pelletier

Altenbuchner

1995

Microbiology

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“…Diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, which irreversibly and specifically react with active site serine residues (32,33), partially inhibited protease IV activity. TLCK totally abolished the activity of protease IV, suggesting the presence of a histidine residue in the active site of the enzyme (34).…”

Section: Resultsmentioning

confidence: 99%

Protease IV, a Unique Extracellular Protease and Virulence Factor from Pseudomonas aeruginosa

Engel¹,

Hill²,

Caballero³

et al. 1998

Journal of Biological Chemistry

156

167

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Comparisons of virulence between a Pseudomonas parent strain and an isogenic mutant devoid of protease IV have demonstrated a significant role for this enzyme during infection. We have characterized purified Pseudomonas aeruginosa protease IV in terms of its biochemical and enzymatic properties, and found it to be a unique extracellular protease. The N-terminal decapeptide sequence of protease IV is not homologous with any published protein sequence. Protease IV has a molecular mass of 26 kDa, an isoelectric point of 8.70, and optimum enzymatic activity at pH 10.0 and 45°C. Purified protease IV demonstrates activity for the carboxyl side of lysine-containing peptides and can digest a number of biologically important proteins, including immunoglobulin, complement components, fibrinogen, and plasminogen. Protease IV is not inhibited by thiol-, carboxyl-, or metalloproteinase inhibitors. The total loss of enzyme activity in the presence of N-p-tosyl-L-chloromethyl ketone and the partial inhibition of enzyme activity by diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride imply that protease IV is a serine protease. Inhibition by dithiothreitol and ␤-mercaptoethanol suggests that intramolecular disulfide bonds are essential for enzyme activity. The characteristics of this enzyme suggest that inhibitors of serine proteases could be developed into a medication designed to arrest tissue damage during Pseudomonas infection.

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“…Kinetics of Inhibition-The inactivation of PPT1 was assumed to proceed via formation of an intermediate Michaelis complex as described previously (11). The reaction is assumed to follow the course shown in Equation 1.…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Das¹,

Bellizzi²,

Tandel³

et al. 2000

Journal of Biological Chemistry

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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 M (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 Å 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.Palmitoyl-protein thioesterase-1 (PPT1) 1 is a newly described lysosomal hydrolase that removes long chain fatty acids from lipid-modified cysteine residues in fatty acylated proteins (reviewed in Ref. 1). Deficiency of the enzyme leads to a lysosomal storage disease, infantile neuronal ceroid lipofuscinosis, which causes blindness, seizures, and cortical atrophy of the brain (2). Lysosomal inclusion bodies, termed granular osmiophilic deposits, accumulate in all tissues, and resemble lipofuscin deposits that occur during normal aging. In keeping with this important lipid-metabolizing role, PPT1 is one of the most abundant lysosomal enzymes in the brain (3). In contrast to most hydrolytic enzymes (such as proteases, lipases, esterases, and thioesterases), PPT1 is insensitive to the serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) (4). The insensitivity of PPT1 to these reagents was an important feature of PPT1 because it allowed for the identification of PPT activity in the presence of contaminating lipase and protease activities. We wondered whether there might be structural and/or mechanistic differences between PPT1 and these other lipolytic enzymes that would account for this observation. We have recently determined the three-dimensional crystal structure of PPT1 in the presence and absence of palmitoyl-CoA and shown that palmitate modifies serine 115 of the enzyme (5), confirming that a serine residue is the catalytic nucleophile.In the current study, we show that although PPT1 is insensitive to inactivation by the ser...

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Sulfonyl Fluorides as Inhibitors of Esterases. II. Formation and Reactions of Phenylmethanesulfonyl α-Chymotrypsin^*

Cited by 295 publications

References 27 publications

A bacterial esterase is homologous with nonhaem haloperoxidases and displays brominating activity

A bacterial esterase is homologous with nonhaem haloperoxidases and displays brominating activity

Protease IV, a Unique Extracellular Protease and Virulence Factor from Pseudomonas aeruginosa

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

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Sulfonyl Fluorides as Inhibitors of Esterases. II. Formation and Reactions of Phenylmethanesulfonyl α-Chymotrypsin*

Cited by 295 publications

References 27 publications

A bacterial esterase is homologous with nonhaem haloperoxidases and displays brominating activity

A bacterial esterase is homologous with nonhaem haloperoxidases and displays brominating activity

Protease IV, a Unique Extracellular Protease and Virulence Factor from Pseudomonas aeruginosa

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

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Product

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Sulfonyl Fluorides as Inhibitors of Esterases. II. Formation and Reactions of Phenylmethanesulfonyl α-Chymotrypsin^*