Tosyl-L-phenylalanine chloromethyl

Tosyl-L-phenylalanine chloromethyl ketone (TPCK) specifically inhibits chymotrypsin by covalently labeling His ". 

This ceramide-mediated apoptosis was shown to be inhibited by the simultaneous addition of PKC activators (Ni et at, 1994 Obeid et al, 1993), implying that PS may activate the ceramide-mediated apoptotic pathway. However, the inhibitors of interleukin-1 converting enzyme (ICE)-like proteases (Caspase), such as tosyl-L-lysine chloromethyl ketone (TLCK), and tosyl-L-phenylalanine chloromethyl ketone (TPCK) which inhibit ceramide-mediated apoptosis, had no effect on PS-induced apoptosis (Figure 4). Thus, PS-induced apoptotic pathway appears to be distinct from that mediated by ceramide. Further studies are required to clarify the molecular mechanisms underlying the PS-induced apoptosis. 

TMCS trimethylchlorosilane imidazole TMG tetramethylguanidine TMP 1,1,3,3-tetramethoxypropane TMS trimethylsilyl TNBS trinitrobenzenesulfonic acid TOCSY total correlation spectroscopy TPA texture profile analysis TPCK Af-tosyl-L-phenylalanine chloromethyl ketone... 

Fig. 10.5. Notable examples of Rcelp inhibitors. Abbreviations shown refer to At,-tosyl-l-phenylalanine chloromethyl ketone (TPCK), p-hydroxymercnribenzoic acid (pHMB), p-hydroxymercuriphenylsulfonic acid (pHMS), mersalyl acid (MSA), (acyloxy)methyl ketone (AOMK), and National Service Center (NSC).

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They are thus more specific for the enzyme active site than are group-specific reagents. Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analog for chymotrypsin (Figure 8.21). TPCK binds at the active site and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme. The compound 3-bromoacetol is an affinity label for the enzyme triose phosphate isomerase (TIM). It mimics the normal substrate, dihydroxyacetone phosphate, by binding at the active site then it covalently modifies the enzyme such that the enzyme is irreversibly inhibited (Figure 8.22). 

A) Tosyl-L-phenylalanine chloromethyl ketone (TPCK) is a reactive analog of the normal substrate for the enzyme chymotrypsin. (B) TPCK binds at the active site of chymotrypsin and modifies an essential histidine residue. 

Histidine 57 is another critical amino acid residue in chymotrypsin. Chemical labeling again provides the evidence for involvement of this residue in the activity of chymotrypsin. In this case, the reagent used to label the critical amino acid residue is A -tosylamido-L-phenylethyl chloromethyl ketone (TPCK), also called tosyl-L-phenylalanine chloromethyl ketone. The phenylalanine moiety is bound to the enzyme because of the specificity for aromatic... 

Lysis is carried out in a disrupting device such as a French pressure cell. The preferred lysis buffer contains 100 mM Tris-HCl, pH 8.0 at 25°C, 0.5 M NaCl, 10 toM 2-mercaptoethanol, 50 xg/ml of phenylmethylsulfonyl fluoride (PMSF), 50 p,g/ml of iV-tosyl-L-phenylalanine chloromethyl ketone (TPCK), 50 (ig/ml of IV-a-tosyl-L-lysine chloromethyl ketone (TLCK), 50 (Jig/ml of pepstatin A, 50 p.g/ml of leupeptin, and 1 mM benzamidine. Typically, the cell sample is thawed in a water bath at room temperature in 1 ml of lysis buffer per 2 g of cells, and passed through a French pressure cell at 16,000 psi to produce a lysate. 

The nomenclature used in this chapter for amino acid and peptide derivatives conforms to the 1971 Recommendations of the lUPAC-IUB Commission on Biochemical Nomenclature, Biochemistry 11, 1726 (1972). Thus the chloromethyl ketone derived from tosyl-L-phenylalanine will be abbreviated as Tos-PheCHCl instead of the more commonly used TPCK. The author recommends the use of systematic abbreviations such as Tos-PheCBtCl since they define the compound concisely, show the relationship between several related halomethyl ketones, and lead to less confusion for those not familiar with shorter abbreviations. 

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