[Substrate specificity of the serine proteinase from Bacillus subtilis, strain 72].
A comparative study of the hydrolysis of various p-nitroanilide substrates (Z-A2-A1-pNA, Z-A3-A2-A1-pNA, and Z-A4-A3-A2-A1-pNA, where A1-An are various amino acid residues, Z is the benzoyloxycarbonylic group and pNA is the p-nitroanilide group), catalyzed by serine proteinase from Bacillus subtilis strain 72, was carried out. It was found that depending on the substrate structure, the hydrolysis may involve both the peptide-p-nitroaniline and the amino acid-amino acid bonds. A kinetic analysis of substrate hydrolysis occurring simultaneously at these two bonds was carried out. The physico-chemical meaning of the kinetic parameters of the given scheme was determined. The quantitative estimation of the enzyme specificity with respect to both hydrolyzing bonds can be found by using the parameters calculated during the analysis of the kinetic curve of p-nitroaniline production. It was found that according to their specificity the amino acid residues at position A1 can be arranged in the following order: L-Leu greater than P-Phe greater than L-Ile greater than L-Ala. The beta-branched amino acid residues, L-Val and L-Ile, do not bind to subsite S1. If these residues occupy position A1, the substrate splitting occurs exclusively between residues A1 and A2. The tetrapeptide N-protected p-nitroanilide substrates are also hydrolyzed at this bond. Partial hydrolysis of the amino acid-amino acid bond between residues A1 and A2 occurs in two cases: i) when residue A1 is loosely bound to subsite S1 and/or, ii) when residue A2 is firmly bound to subsite S1.
Gololobov
M Iu
MIu
Morozova
I P
IP
Voiushina
T L
TL
Timokhina
E A
EA
Stepanov
V M
VM
D000596:Amino Acids / Q000032:analysis; D000814:Aniline Compounds / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*
D000596:Amino Acids; D000814:Aniline Compounds; C008625:nitroaniline; D013381:Subtilisins
Substratnaia spetsifichnost' serinovoĭ proteinazy Bacillus subtilis, sht. 72.