Active Site Labelling

However, (26) should bind strongly to class II aldolases on account of its chelating ability and this has been found to be the case with rabbit muscle fructose diphosphate aldolase where (26) functions as a competitive inhibitor. Triose phosphate isomerase is also strongly inhibited by (26) this may be due to its similarity to the cw-eriediolate (28), which is an intermediate in the reaction pathway of this enzyme. Acyldihydroxyacetone phosphates are important intermediates in the biosynthesis of glycerolipids and the acyl 

Saunders, Some Aspects of the Chemistry and Toxic Action of Organic Compounds containing Phosphorous and Fluorine , Cambridge University Press, Cambridge, 1957. 

The chemical shift of the phosphorus resonance of various nucleotides has been studied as a function of pH in the presence and absence of RNase A. The signal shifts upheld on protonation of the phosphate and the apparent pATa of the phosphate group in 2 -CMP complex with RNase is the same as the pATa of histidine-119 in this enzyme as determined by n.m.r. From n.m.r. relaxation rates for the ternary complex manganese(n)-phosphate-E. coli alkaline phosphatase, it has been concluded that an outer-sphere complex is formed which has a shorter lifetime than the enzyme turnover rate. The latter conclusion is consistent with the participation of the complex in the enzymic reaction. 

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Zhang, Z.-Y., Dixon, J. E. Active site labeling of the yersinia protein tyrosine phosphatase The determination of the pKa of active site cysteine and the function of the conserved histidine 402. Biochem. 32 (1993) 9340-9345. 

Treatment of 9-(/ -D-ribofuranosyluronic acid)adenine with diphenylphosphoro-chloridate and orthophosphate or tripolyphosphate yields (62) and (63), which, although unstable, inhibit rabbit AMP aminohydrolase and pyruvate kinase, respectively, with behaviour characteristic of active-site-specific reagents.98 Adenylate kinases from several sources are inactivated by iV6-[2- and 4-fluorobenzoyl]-adenosine-5 -triphosphates, with kinetics characteristic of active-site labelling, although these compounds were without effect on yeast hexokinase and rabbit pyruvate kinase.99... 

M. K. Johnson, P. Glynn, Active-Site Labelling and Purification of Neuropathy Target Esterase (NTE) Using a Biotinylated Organophosphorous Compound , Toxicologist 1993, 73,211. 

The level of display is the average number of enzymes displayed per phage particle. For a wild-type enzyme whose activity is not affected by the phage environment, the level of display is evaluated by dividing the kC3t of the phage by the /ccat of the free enzyme. Otherwise, it can be evaluated by Western blot or, when possible, by active-site labeling. 

From active site labelling experiments and from sequence comparison of NRs with homologous proteins, functionally important residues can be predicted and localised on the NR sequences. 

Pitscheider M, Sieber SA (2009) Cinnamic aldehyde derived probes for the active site labeling of pathogenesis associated enzymes. Chem Commun 2009 3741-3743... 

Bottcher T, Sieber SA (2008) P-Lactones as privileged structures for the active-site labelling of versatile bacterial enzyme classes. Angew Chem Int Ed 47 4600 -603... 

Figure 11 Mechanism-based probes for cysteine protease active site labeling, (a) E-64-mediated inhibition of cysteine proteases via nucleophilic attack of the catalytic cysteine at the epoxy group, (b) E-64-based probe with biotin reporter tag and tyrosine as 125l iodination site for radioactive labeling, (c) Acyloxymethylketone probe for the complementary labeling of cysteine proteases.

Figure 19 Mechanism of /3-glycosidase active site labeling with probes for (a) retaining and (b) inverting /3-glycosidases.

Figure 21 Haloacetamide-derived probes as potent tools for the labeling of arginine deaminase PAD4. The probe design is based on arginine as a natural substrate that is converted into a F/CI reactive group. Active site labeling occurs via a nucleophilic substitution of the halogene by the catalytically active cysteine of PAD4.

Pot DA, Dixon JE (1992) Active site labeling of a receptor-like protein tyrosine phosphatase. J Biol Chem 267 140-143... 

Figure 2. Active-site labelling of rabbit muscle aldolase (1)...

The absolute number of essential residues, either of the same or different types of amino acids, can frequently be determined. One of the simplest and best methods is the use of one of the several types of active-site labeling reagents which have an easily monitored group attached. Such a group could be labeled with a radioactive element or with a distinct optical absorption band. One that has frequently been used with the serine -type enzymes is diisopropylfluorophosphate labeled with 32P. 

Later, the presence of topa quinone was accordingly confirmed in the amine oxidases from porcine serum and kidney and pea seedling by resonance Raman spectrometry of active-site labeled peptides [48]. Comparison of amino acid sequences of these peptides with the sequences of those from bovine plasma and H. polymorpha amine oxidases demonstrated the presence of a consensus sequence Asp-TPQ-Asp/Glu as shown in Fig. (1). Using the pH-dependent shift of the absorption maximum of the enzyme p-nitrophenylhydrazone, which is considered to be a reliable indirect proof, the presence of topa quinone was also shown... 

Ester and sulphonium salt formation in the active-site labelling of jS-D-galacto-sidase from E. coli by 2,6-anhydro-l-deoxy-l-diazo-D- /ycera-L-manno-heptitol has been examined.2,6-Anhydro-l-deoxy-l-diazo-D- /yccro-L-wflnno-heptitol... 

TABLE I Examples op Active-Site Labeling with Bbomopybovate ... 

Active-Site Labeling of Thymidylate Synthetase with 5-Fluoro-2 -deoxyuridylatei... 

Active-Site Labeling with Conduritol B Epoxide or Its... 

Bromoadenosine monophosphate, 99 3-Bromo-2-butanone-l,4-diol diethyl ketal, synthesis of, 393, 394 Bromocolchicine, 567-571 binding to brain extracts, 570,571 synthesis of, 569 Bromoconduritol, 369,370 inhibition of glucosidase, 381 synthesis of, 380, 381 a-Bromo-ft>-(3-cyanophenyl)alkane, 122 6-Bromo-6-deo conduritol B epoxide, active site labeling, 377-380 3-Bromo-l,4-dihydroxy-2-butanone 1,4-bisphosphate, 142, 392, 396, 397 synthesis of, 392-395... 

Competitive inhibition, 4 Concanavalin A, 85,368 binding site, 75 Conduritol B, 369 synthesis of, 373 Conduritol B epoxide, 369 active site labeling, 377-380 labeling with, 370 reaction with /3-glucosidases, 369 synthesis of, 371-376 Conduritol B tetraacetate, synthesis of, 373... 

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