Activation and stabilization of alpha-chymotrypsin by cationic additives.
alpha-Chymotrypsin activity was tested with N-glutaryl-l-phenylalanine p-nitroanilide (GPNA) in aqueous media in the presence of synthetic surfactants, which differ in the flexibility of their bulky head groups. Superactivity can be ascribed to the presence of the tributylammonium residue on the surfactant head group, as in p-octyloxybenzyltributylammonium bromide (pOOTBABr), while in the presence of a more rigid moiety, i.e. a cyclic one, no activation was found. A nonmicellizable quaternary ammonium salt, the tetrabutylammonium bromide (TBABr), which has a head group structure very similar to pOOTBABr, not only induces a remarkable superactivation, at a concentration 80-fold higher than pOOTBABr, but also allows the enzyme to retain a high residual activity for long periods of time. The presence of a lipophilic chain, which by interacting with apolar residues on the enzyme surface, probably penetrates into hydrophobic pockets of the protein and causes a rapid inactivation. In 0.4 m TBABr, a 20-fold increase both in kcat and Km values, with respect to buffer alone, was found. The increase of Km could be attributed either to a true decrease in affinity between enzyme and substrate or alternatively to the presence of TBA+ ions near the catalytic region. They could interact with protein residues around the active site and bind to negatively charged GPNA molecules, lowering the local substrate concentration. Spectroscopic experiments (CD and fluorescence) show minor changes of protein conformation in 0.4 m TBABr, while at 1 m a strong modification of both spectra was observed.
Spreti
N
N
Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università dell'Aquila, Italia.
Di Profio
P
P
Marte
L
L
Bufali
S
S
Brinchi
L
L
Savelli
G
G
D000818:Animals; D002412:Cations; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000494:pharmacology
D002412:Cations; D013501:Surface-Active Agents; D002918:Chymotrypsin; C117608:alpha-chymotrypsin