Thermolysin: Kinetic Study with Oligopeptides

Abstract

Thermolysin is a well-known protease which exhibits its specificity against hydrophobic amino acid residues such as L-leucine, L-phenylalanine, etc. whose amino groups donate the susceptible peptide bonds (amino-endopeptidase). The present study was undertaken to investigate the effects of neighboring residues surrounding the sensitive amino acid residues at the amino-side in peptide substrates. For the purpose, a kinetic study was made using various synthetic oligopeptides such as Z-A-(Gly),-i;Leu-Ala or Z-Gly+eu-(Gly),-B (A or B = various I)-or L-amino acid residues; n = 0, 1 and/or 2; the arrow shows the bond split) as substrates. Other kinetic or inhibition studies were also made. These studies indicated that the specificity is affected by a t least three amino acid residues on the N-terminal side and by two amino acid residues on C-terminal side from the sensitive amino acid residue (at amino-side) in peptide substrates. The effect of each of the five neighboring amino acid residues for appearance of the specifiicity was similar with that of the corresponding one which had been observed in a neutral protease of Bacillus subtilis, but that was not completely the same.Thermolysin is a thermo-stable protease produced by Bacillus thermopotwlyticus. It was discovered by Endo El], and its enzymatic specificity was first studied by Matsubara et al.[2], using beef cytochrome c as substrate, who assumed that the enzyme is specific for peptide bonds containing the amino group of hydrophobic amino acid residues such as L-leucine, L-isoleucine, L-phenylalanine, etc. This assumption was supported by a further study by the same authors using insulin using Z-Gly-X-NH, (X = various amino acid residues of D-or L-configuration; the arrow shows the bond split) as substrates indicated that the highest activity was obtained when X was L-leucine, or L-phenylalanine, well reflecting the specificity against large moleular peptides or proteins. These characteristics of specificity have since been found to be Unueuul Abbreviations. DFP, diisopropyl phosphofluoridate ; Z, benzyloxyoarbonyl.A 181. The specificities of various neutral proteases from bacterial origin, including thermolysin, were shown [15] to be affected by at least the next five residues adjacent to the sensitive amino acid residue in peptide substrates. A further study of B. subtilis neutral protease [16,17] with various synthetic peptides somewhat clarified the contribution to specificity of each of these five amino acid residues in peptide substrates. The present kinetic study of thermolysin with synthetic peptides was undertaken to compare the influences of neighboring residues on the specificity against the amino acid residue donating the amino group to be hydrolyzed with those found with the B. subtilis enzyme.

Hydrophobic interaction in thermolysin specificity

“…Neutral metalloendopeptidase thermolysin (EC 3.4.24.4) catalyzes the hydrolysis of such peptide bonds where the amino side of the bond is contributed preferably by an amino acid with a large hydrophobic residue, e.g., Phe or Leu [1][2][3], which points to the presence of a corresponding hydrophobic pocket in the active site of the enzyme. This conclusion is in accordance with X-ray crystallography data [4].…”

Cyclin B1 interacts with the BH3-only protein Bim and mediates its phosphorylation by Cdk1 during mitosis

“…sites, 39 and the sample was analyzed by mass spectrometry using a Thermo Fisher LTQ-Orbitrap. The peptide sequence LQTEPQDRSPAP of BimL was detected, and a molecular mass gain of 79.97 was detected on Ser44, which is indicative of phosphorylation (Fig.…”

Exploring the subsite‐structure of vimelysin and thermolysin using FRETS‐libraries

“…The substrate specificity of thermolysin and related enzymes has been thoroughly examined through many structural and kinetic studies [6][7][8][9]. The P1 position (immediately N-terminal to the cleavage site, according to the nomenclature of Schechter and Berger) and P1 0 positions are the most important for substrate specificity: Phe is strongly preferred at the P1 position, and Leu or Phe at the P1 0 position.…”