TPCK

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

Figure 7. Two examples of irreversible inactivators that are not suicide substrates a) TPCK, a classic" affinity label of the serine protease chymotrypsin, b) ZFK-CH2-mesitoate, a quiescent" affinity label of the cysteine protease cathepsin B, and c) the kinetic scheme for both forms of affinity label-inactivation.

More specific evidence came from affinity labeling with molecules which could react with specific amino acid group sat or adjacent to the substrate site. These labels were substrate analogues and competitive inhibitors. Substituted aryl alkyl ketones were used. TV-p-toluene-sulphonyl-L-phenylalanine chloromethyl ketone (TPCK) blocked the activity of chymotrypsin. Subsequent sequence analysis identified histidine 57 as its site of binding (see Hess, 1971, p 213, The Enzymes, 3rd ed.). Trypsin, with its preference for basic rather than aromatic residues adjacent to the peptide bond, was not blocked by TPCK but was susceptible to iV-p-toluenesulphonyl-L-lysine chloromethyl ketone (TLCK) (Keil, ibid, p249). 

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. 

In consideration of the close resemblance of the inhibitor to a natural substrate for the enzyme, and of the enzymic process of inhibition, the reaction of the enzyme with TPCK has been described as affinity labelling. (Schoellman and Shaw, 1962, 1963). Many other examples of affinity labelling have subsequently been discovered. 

The principles of the method are very nicely illustrated by one of the first affinity labeling experiments, the reaction of /exs-i.-phenylalanine chloro-methyl ketone (TPCK) with chymotrypsin.1 TPCK resembles substrates like fexs-L-pheny 1 alanine methyl ester, but the chloromethyl ketone group of TPCK is an alkylating reagent. 

Halomethyl ketones and acids are known to react with thiols and imidazoles. TPCK reacts far more rapidly with chymotrypsin than it does with normal histidine-containing peptides because of its high reactivity as an affinity label. This can be seen in Table 9.2 for an analogous chloromethyl ketone. In addition to this important diagnostic feature, the irreversible inhibition of chymotrypsin by TPCK has four other characteristic features 1,4... 

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

TPCK-treated trypsin, TLCK-treated chymotrypsin, and endoproteinase-Glu-C (Staphlococcal V8 protease) are all obtainable from Sigma (Poole, UK)... 

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... 

Competitive inhibition of the carboxypeptidase from A. saitoi by small substrates was found with hydrocinnamic acid, indole-3-propionic acid, and 4-phenylbutyric acid [80], The K for hydrocinnamic acid inhibition was 4 x 10 4 M. Diisopropylfluorophosphate (DFP) and tosyl-L-phenylalanylchloromethane (TPCK) were also powerful inhibitors of the carboxypeptidase from A. oryzae (80). />-Chloromercuribenzoate (PCMB) and iodoacetic acid were also powerful inhibitors of the carboxypeptidase from A. saitoi, while the inhibitors of DFP, TPCK, PCMB, and iodoacetic acid on the carboxypeptidase from A. saitoi were less than that of A. oryzae [80], As the carboxypeptidase activity of A. saitoi has no effect when used with ethylenediaminetetraacetate (EDTA) and o-phenanthroline, the enzyme is a different type of carboxypeptidase from those of the pancreatic sources, carboxypeptidase A and carboxypeptidase B [80],... 

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