Enhancement of alpha-chymotrypsin-catalyzed hydrolysis of specific p-nitroanilide substrates by 4-phenylbutylamine derivative of hen egg-white lysozyme.
Modification of hen egg-white lysozyme by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in presence of 4-phenylbutylamine yielded derivatives, which contained 0.6--0.7 modified residues and retained about 60% of the original activity. Kinetic studies revealed that the modified-lysozyme increases approx. 20-fold the kcat of hydrolysis of SucGly2Phe-4-nitroanilide by alphachymotrypsin, without changing the Km. The apparent dissociation constant of phenylbutylamine-modified lysozyme . chymotrypsin complex was found to be 0.03 mM and independent of substrate concentration. The accelerating effect of the modified lysozyme was also observed with other p-nitroanilide substrates of alpha-chymotrypsin. However, the hydrolysis of other substrates, acylation by active site titrant or inhibition by irreversible or competitive inhibitors were uneffected. The enhancing effect of the modified lysozyme seems to be very specific since other chymotrypsin-like enzymes, or serine proteinases except delta-chymotrypsin, were not influenced and phenylbutylamine derivatives of alpha-lactalbumin or ribonuclease were lacking any enhancing effect. Smaller, but significant enhancing effect was found also in lysozyme substituted by benzylamine, beta-phenylethylamine and tryptamine and in inactive derivatives of lysozyme substituted by phenylbutylamine. Competitive inhibitors of lysozyme such as N-acetyl-D-glucose amine oligomers, (GlcNAc)2 and (GlcNAc)3 abolished partially the accelerating effect of phenylbutylamine-modified lysozyme, indicating that the substituted group is located in the vicinity of the binding site.
D000813:Anilides; D002082:Butylamines; D002918:Chymotrypsin / Q000378:metabolism*; D004529:Egg White; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000813:Anilides; D002082:Butylamines; D010649:Phenylalanine; C018389:N-succinylglycyl-phenylalanine-4-nitroanilide; D009113:Muramidase; D002918:Chymotrypsin