Arazoformyl peptide surrogates as spectrophotometric kinetic assay substrates for carboxypeptidase A.
N-(4-Methoxyphenylazoformyl)-L-phenylalanine is efficiently cleaved by the enzyme bovine carboxypeptidase A into fragments anisole, molecular nitrogen, carbonate, and phenylalanine, in the course of which an intense spectral absorption of the substrate (epsilon350 = 19,000 M-1 cm-1) disappears completely. This furnishes a sensitive spectrophotometric detection of the protease. Steady-state catalytic velocity depends upon enzyme and substrate concentrations in the normal manner, and a Michaelis-Menten Km value of 0.11 mM and a kcat value of 44 s-1 were measured at pH 7.5 in saline solution. These parameters have a pH dependence typical for the enzyme. With saturating amounts of substrate, a solution containing 10 nM enzyme produces a spectral absorptivity change at 350 nm of -0.03 a.u./min (1-mm pathlength), suitable for assay purposes. Catalysis may alternatively be monitored at wavelengths as long as 400 nm.
Mock
W L
WL
Department of Chemistry, University of Illinois at Chicago, 60607-7061, USA.
Liu
Y
Y
Stanford
D J
DJ
D000818:Animals; D000840:Anisoles / Q000378:metabolism; D001391:Azo Compounds / Q000378:metabolism*; D002254:Carbonates / Q000378:metabolism; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D002417:Cattle; D005470:Fluorometry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives* / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D011199:Potentiometry; D013053:Spectrophotometry
D000840:Anisoles; D001391:Azo Compounds; D002254:Carbonates; C102220:N-(4-methoxyphenylazoformyl)leucine; C102219:N-(4-methoxyphenylazoformyl)phenylalanine; C102221:N-(phenylazoformyl)phenylalanine; D010649:Phenylalanine; C044911:N-(3-(2-furyl)acryloyl)phenylalanine; C060998:anisole; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D007930:Leucine; D009584:Nitrogen