Fluorogenic substrate hydrolysis

The fluorogenic hydroxyketone 14 discussed above (Scheme 1.4) can also be used to form esters such as 26 which can be used as fluorogenic substrates for lipases [37]. In this case, however, the esters are quite unstable despite being ali-phahc alcohol esters. The relatively rapid spontaneous hydrolysis in this case is probably due to an assisted mechanism involving acyl transfer in the hydrated form of the ketone. 

The CUps-O epoxide 33-41 and related substrates are currently the only available selective fluorogenic substrates for EH. On the other hand various indirect assays have been reported to detect either the unreacted epoxide [46] or the carbonyl product resulting from periodate cleavage of the 1,2-diol product [26,47, 48], These assays are suitable for fluorescence or colorimetric assays for the hydrolysis of any epoxide of interest... 

Arylsulfatase activity is usually measured with the chromogenic substrate 4-nitrocatecholsulfate. (A fluorogenic substrate, 4-methylumbelli-ferylsulfate, is also available but is less frequently used because it is hydrolyzed only very slowly.) The assay system of Baum et al. (1959), which was developed to permit the determination of arylsulfatase A in the presence of the B isoenzyme, is still most widely used. The unusual time course of the hydrolysis of 4-nitrocatecholsulfate by arylsulfatase A (Roy, 1953 Baum et al., 1958 Stinshoff, 1972) may present a problem for standardization of the enzyme preparation and should be taken into account, such as by using short incubation times (15-30 min). The enzyme unit is usually defined as the amount of enzyme that hydrolyzes 1 p.mol of this substrate per minute. 

Figure 5. Hydrolysis of fluorogenic substrate 1 by HTV protease at 37 C as monitored by steady state fluorescence spectroscopy (Xex+340, A m=490). The reaction was carried out with 10 pM substrate at pH 4.7 in a buffer solution containing 0.1 M NaOAc, 1.0 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% DMSO and 1 mg/mL bovine serum albumin. The arrow indicates the point of addition of HTV protease to a final concentration of 35 nM. Product analysis was carried out by HPLC, mass spectrum and fluorescence lifetime. Inset The initial phase of the hychx)lysis reaction used for rate determinations.

How does the GAr interfere with ubiquitin/proteasome-dependent proteolysis In principle, any of the three identified steps in the pathway, recognition, targeting, or proteolysis, may be affected. However, the strictly r/ s-acting effect of the domain speaks against a direct interference with the enzymatic activity of the 20S particle. Indeed, addition of synthetic glycine-alanine peptides did not affect the hydrolysis of fluorogenic substrates by purified 20S proteasomes (A. Leonchiks, A. Sharipo,... 

When a glycoside is present in a solid selection medium fluorescent zones appear under UV light around points of inoculation or around colonies of cells secreting the corresponding glycosidase. Umb-L as an example of a fluorogenic substrate, and products of its hydrolysis are shown in Figure 2. 

Methods of preparing highly purified jS-o-acetamidodeoxyhexosidases A and B from human placenta have been described. The purified isoenzymes possess identical molecular weights, exhibit similar kinetics with synthetic fluorogenic substrates, and catalyse the hydrolysis of Tay-Sachs ganglioside and the asialo-derivative thereof. Although both isoenzymes consist of four subunits (each of... 

Table 7. Kinetic parameters for hydrolysis of fluorogenic peptide substrate Boc-Leu- ...

Most of the early tests were based on the hydrolysis of the natural sphingolipid which had been labelled in the hydrophilic portion. Thus, the substrate and products could be easily separated by two-phase solvent partition or by precipitation of the product. In many cases artificial chromogenic or fluorogenic glycosides could be used instead of radiolabelled substrates. However, in these cases it is very important to demonstrate that the conditions and specificities of the enzynie reactions are such that the results can be extrapolated to the natural substrate (e.g. Peters et al., 1975). Thus, in the case of Niemann-Pick or Krabbe s diseases the unsubstituted... 

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