N-anthraniloyl-Ala-Ala-Phe-4-nitroanilide, a highly sensitive substrate for subtilisins.
Arch Biochem Biophys
; 294(2): 703-6, 1992 May 01.
Article
em En
| MEDLINE
| ID: mdl-1567226
ABSTRACT
A new substrate for subtilisins, anthraniloyl-Ala-Ala-Phe-4-nitroanilide, has been synthesized and characterized. The peptide is a fluorogenic substrate that is intramolecularly quenched without loss of its chromogenic properties and offers a possibility for double-assay kinetic analysis. The kinetic parameters determined for subtilisin Carlsberg are Km = 0.004 mM, kcat = 104 s-1, and those for subtilisin BPN' are Km = 0.020 mM, kcat = 49 s-1. The substrate is extremely sensitive for subtilisins; the specificity constants are 10-fold higher than the corresponding values for the widely used substrate, succinyl-Ala-Ala-Pro-Phe-4-nitroanilide, and 200- to 1000-fold higher than the values obtained with succinyl-Ala-Ala-Phe-4-nitroanilide. The favorable effect of the anthraniloyl group as a P4 residue in the substrate sequence Ala-Ala-Phe-4-nitroanilide was assumed to be due to an ability to stiffen S4-P4 interactions. The mechanism proposed is hydrogen bond formation between the phenol group of tyrosine-104 and the amino group of the anthraniloyl moiety. In the spectrophotometric assay with the new substrate, the lower detection limit for subtilisin Carlsberg was 1 nM.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligopeptídeos
/
Subtilisinas
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Arch Biochem Biophys
Ano de publicação:
1992
Tipo de documento:
Article