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A phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases.

ABSTRACT: Mca-Gly-Asp-Ala-Glu-Tyr(PO(3)H(2))-Ala- Ala-Lys(DNP)-Arg-NH(2), where Mca is (7-methoxycoumarin-4-yl)acetyl and DNP is 2,4-dinitrophenyl, was synthesized as a fluorogenic substrate for protein tyrosine phosphatases (PTPs). In the peptide, the fluorescent Mca group is quenched efficiently by the DNP group. Although the fluorescence intensity of the substrate was practically unchanged upon PTP-catalysed dephosphorylation, it increased approx. 120-fold upon subsequent treatment with chymotrypsin. Analysis by HPLC showed that chymotrypsin cleaved only the dephosphorylated substrate at the Tyr-Ala bond. Thus with the aid of chymotrypsin, dephosphorylation of the substrate can be measured fluorometrically. A strictly linear correlation was observed between PTP concentration and dephosphorylation rate. The fluorogenic substrate was dephosphorylated by some PTPs much more rapidly than the corresponding (32)P-labelled substrate used for comparison, whereas alkaline phosphatase dephosphorylated the two substrates at similar rates. The fluorogenic substrate is therefore more specific for PTPs than the radiolabelled substrate. The assay with the fluorogenic substrate could be applied to the estimation of kinetc parameters and measurement of PTP activity in crude-enzyme preparations. The lower detection limit of our assay (1 microM substrate in 200 microliter of reaction mixture) was estimated to be 0.2-0.4 pmol, whereas it was estimated to be about 1 pmol in the assay that used (32)P-labelled peptide (specific radioactivity of approx. 1000 c.p.m. /pmol). Our assay is simple, specific, highly sensitive and non-radioisotopic, and hence would contribute greatly to the development of PTP biology.

SUBMITTER: Nishikata M

PROVIDER: S-EPMC1220565 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Project description:The application of D-stereospecific proteases (DSPs) in resolution of racemic amino acids and in the semisynthesis of proteins has been a successful strategy. The main limitation for a broader application is, however, the accessibility of suitable DSPs covering multiple substrate specificities. To identify DSPs with novel primary substrate preferences, a fast specificity screening method using the easily accessible internally quenched fluorogenic substrate aminobenzoyl-D-arginyl-D-alanyl-p-nitroanilide was developed. By monitoring both UV/vis-absorbance and fluorescence signals at the same time it allows to detect two distinct D-amino acid substrate specificities simultaneously and separately with respect to the individual specificities. In order to identify novel DSP specificities for synthesis applications, DSPs specific for D-arginine were of special interest due to their potential ability as catalysts for substrate mimetics-mediated peptide and protein ligations. D-alanine in the substrate served as positive control and reference based on its known acceptance by numerous DSPs. In silico analysis suggested that DSPs are predominantly present in gram-positive microorganisms, therefore this study focused on the bacilli strains Bacillus thuringiensis and Bacillus subtilis as potential hosts of D-Arg-specific DSPs. While protease activities toward D-alanine were found in both organisms, a novel and so far unknown D-arginine specific DSP was detected within the culture supernatant of B. thuringiensis. Enrichment of this activity via cation exchange and size exclusion chromatography allowed isolation and further characterization of this novel enzyme consisting of a molecular mass of 37.7 kDa and an enzymatic activity of 8.3 U mg-1 for cleaving the D-Arg|D-Ala bond in the detecting substrate. Independent experiments also showed that the identified enzyme shows similarities to the class of penicillin binding proteins. In future applications this enzyme will be a promising starting point for the development of novel strategies for the semisynthesis of all-L-proteins.

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A phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases.

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