N-Succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin

Herein the reactivity of the enzyme α-chymotrypsin in the confinement of polystyrene-block-poly(acrylic acid) (PS-b-PAA) vesicles was investigated. Enzyme and substrate molecules were encapsulated in PS-b-PAA vesicles with internal diameters ranging from 26 nm to 165 nm during the formation of the vesicles. While the loading efficiencies of enzyme and substrate molecules were practically identical for vesicles of identical size, they were found to increase with decreasing vesicle size. The kinetics of the α-chymotrypsin catalyzed hydrolysis of N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) was evaluated following the increase of the absorption of the product 7-amino-4-methylcoumarin by UV/Vis spectroscopy. The values of the catalytic turnover number obtained for reactions inside vesicles with different sizes showed an increase of up to fourteen times compared to the bulk value with decreasing vesicle volume, while the values of the Michaelis-Menten constant decreased, respectively. This increase in reactivity of α-chymotrypsin is attributed to the effect of vesicle-wall interactions in the finite encapsulated space, where the reagents could diffuse, leading to enhanced collision frequencies.

A soluble tripeptidylaminopeptidase has been isolated from human post-mortem cerebral cortex by anion exchange, hydrophobic interaction and size-exclusion chromatography. From gel filtration studies the active enzyme can exist in both high molecular weight (M(r) > 10(6) and smaller forms. The enzyme hydrolyses Ala-Ala-Phe-7-amido-4-methylcoumarin with a pH optimum of around 7.5 and Km of 148 microM. It did not hydrolyse N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, aminoacyl- or dipeptidyl-7-amido-methylcoumarins and was not inhibited by bestatin. The enzyme was inhibited by phenylmethylsulphonyl-fluoride, 3,4-dichloroisocoumarin, N-hydroxymercuriphenyl-sulphonic acid and N-ethylmaleimide showing that its activity is serine and cysteine dependent. The purified enzyme released tripeptides from several naturally occurring neuropeptides with quite broad specificity. Cholecystokinin octapeptide, angiotensin III and neurokinin A were the most rapidly hydrolysed. Peptides with Pro residues around the point of cleavage were not hydrolysed.

Clubroot, caused by Plasmodiophora brassicae, is one of the most serious diseases of cultivated cruciferous crops in the world. However, the basis for pathogenicity in P. brassicae is not well understood. In this study, a serine protease gene (PRO1) was cloned from P. brassicae and its molecular characteristics were investigated. Southern analysis and specific polymerase chain reaction (PCR) amplification indicated that PRO1 is a single-copy gene present in a broad range of P. brassicae pathotypes. Northern analysis revealed that the expression of PRO1 was induced during plant infection, and that the quantity of transcript fluctuated according to the stage of pathogenesis. Amino acid sequence analysis suggested that the encoded protein (Pro1) belongs to the S28 family of proteases, with a predicted signal peptide and a theoretical molecular mass of 49.4 kDa. The open reading frame (ORF) of PRO1 was transferred into Pichia pastoris and Pro1 was heterologously produced. Pro1 showed proteolytic activity on skimmed milk and N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, and the activity could be inhibited by serine protease inhibitors and the chelating agent ethylenediaminetetraacetic acid. The optimal temperature of Pro1 was 25 degrees C, and it exhibited high activity at pH 6.0-6.4. These values coincide with the temperature and pH conditions favourable for P. brassicae resting spore germination in the field. When Pro1 was used to treat canola root exudates, it enhanced the stimulating effect of the root exudates on P. brassicae resting spore germination, indicating that Pro1 may play a role during clubroot pathogenesis by stimulating resting spore germination through its proteolytic activity.