Thermolysin peptides

Yano, S., Suzuki, K., and Funatsu, G. 1996. Isolation from a-zein of thermolysin peptides with angiotensin I-converting enzyme inhibitory activity. Biosci. Biotechnol. Biochem. 60, 661-663. 

Fragmentation into larger peptides retaining intact sequences of arginine residues was achieved by hydrolysis with thermolysin (1/15 M phosphate buffer, pH 8.05, 37 °C, 3 h, substrate/enzyme = 200—300, w/w). The thermolysin peptides obtained with good recovery are also illustrated in Chart VIII-9. The structure and recovery of the thermolysin peptides obtained from iridine I are shown in Table VIII-5 as well as Chart VIII-9. The results revealed the presence of two molecular species (I-Ia and I-Ib) in the iridine fraction (I-I). Some difference between the two species of molecules was also found in the number of arginine residues in their clusters. This observation was further supported by the results of Neutral Protease peptides, as described below. 

Chart VIII-9. The amino acid sequences of salmine AI, iridine I (a) and (b), and iridine II with demonstration of peptides obtained by enzymic hydrolyses. The symbols used are A, Arg T, tryptic peptide Tm, thermolysin peptide NP, Neutral Protease peptide NP-7-NP, redigested Neutral Protease peptide from NP-No. 7 Tm-6-NP, Neutral Protease peptide from Tm-No. 6 (from Ando and Watanabe, 1969)... 

These Neutral Protease peptides from iridine I revealed, along with the thermolysin peptides, the presence of 2 molecular species of the same composition except for substitution of a valine residue with an isoleucine residue between the two. Iridine II ... 

The chemical structures of these peptides were established in the usual way by quantitative amino-acid analysis and sequence analysis by a combination of chemical and enzymic methods, including dinitrophenylation, dansylation, Edman degradation, hydrazinolysis and digestion with leucine aminopeptidase and carboxypeptidases A and B. The Neutral Protease peptides of the salmine and iridine components or derivatives thus identified are listed in Table VIII-6 with their recovery values. Differences in the amino-acid sequences and amounts of the peptides obtained from iridine I aroused the suspicion that two molecular species (a and b) were present in the iridine I component. This result, as already described, was supported by observations on the thermolysin peptides of iridine I. 

Although equihbrium-controUed peptide synthesis has been successfully used on a number of occasions, including thermolysin-catalyzed synthesis of aspartame (126) and semisynthesis of insulin (127), the method has a significant drawback a water-miscible organic cosolvent added to the reaction medium to suppress the ionization of unactivated carboxy components significantly reduces the reaction rate. 

Thermolysin acts simultaneously at several sites on the Ca -ATPase without accumulation of large fragments this property proved useful in the sequence analysis of the Ca -ATPase [78,79,82,83], and in the isolation of SH-group-containing peptides [257]. Small fragments also accumulate after treatment of sarcoplasmic reticulum with subtilisin [256]. 

Fig. 10. (A) Far-ultraviolet CD spectra of fragment 206-316 of thermolysin at different temperatures in 20 raM Tris-HCl buffer, pH 7.2. Numbers near the curves indicate temperature in degrees Celsius. The peptide concentration was 0.2 mg/ml. (B) Temperature dependence of [0]/[0]o at 220 nm of fragment 206-316, where [0]o is the mean residue ellipiticity at 22°C. Reprinted with permission from Peptides Proceedings of the Fifth American Peptide Symposium, John Wiley Sons, Inc., 1977. Copyright by John Wiley Sons, Inc.

Carboxypeptidase A was the first zinc enzyme to yield a three-dimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb etai, 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argosetai, 1978 Kesterand Matthews, 1977 Monzingoand Matthews, 1984 Matthews, 1988 Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.. 

P.A. Bartlett, A. Otake, Fluoroalkenes as peptide isosteres Ground state analog inhibitors of thermolysin, J. Org. Chem. 60 (1995) 3107-3111. 

The potential utility of peptides as therapeutics with clinical applications is limited by its metabolic instability or poor transmembrane mobility. Consequently, the preparation of metabolically stable peptide analogs that can either mimic or block the function of natural peptides or enzymes is an important area of medicinal chemistry research. Synthesis of fluoroolefin amide isosteres, its incorporation in peptidomimetics, and the influence of that isosteric substitution on the inhibition of several enzymes such as peptidyl prolyl isomerases, dipeptidyl peptidase IV, and thermolysin is described. Moreover, protein folding and activity... 

Proteolysis of peptides and proteins by enzymes occurs in a selective or nonselective manner. Chymotrypsin, trypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease, and en-dopeptidase Asp-N are frequently listed as selective enzymes, whereas thermolysin, pepsin, subtilisin, and elastase belong to the nonselective enzymes, although thermolysin preferentially cleaves peptide bonds before hydrophobic residues. 

The helix contents of five peptide fragments from the protein thermolysin have been determined by CD and NMR in both water and 30% TFE. 85 The helix content was obtained from CD by the method of Chen et a I.162 and the NMR method utilized chemical shifts. 84 Four of the five peptides correspond to helical regions in the intact protein, and one corresponds to an Q-loop. 86 The rms difference between CD and NMR helix contents for the five peptides under the two conditions is 7.5%. One peptide shows the largest deviations (0 vs 13% in water, 45 vs 62% in 30% TFE). If it is excluded, the rms deviation decreases to 4%. The peptide showing the largest deviation, residues 258-276 from the thermolysin sequence, has two Tyr and one Phe (with the Phe adjacent to one of the Tyr in the sequence), and therefore it has an above-average content of aromatic amino acids, which can perturb both the CD spectrum and NMR chemical shifts. Of the other four peptides, three have a single aromatic residue and the fourth has two aromatics. 

Lee, T. G. and Maruyama, S. (1998). Isolation of HTV-1 protease-inhibiting peptides from thermolysin hydrolysate of oyster proteins. Biochem. Biophys. Res. Commun. 253, 604-608. 

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