Thermolysin amino acid sequence

Thermolysin is a single-chain protease composed of 316 amino acids its amino acid sequence and three-dimensional structure (Colman et at, 1972) have been determined. The native protein has no disulfide bonds, but does contain both Ca2+ and Zn2+, which strongly stabilize the structure. 

Jornvall and Harris (91) presented data for the structures around all of the 14 cysteine residues in each protein chain. Analysis by Jornvall (92,93) of different peptide mixtures obtained after treatment of the protein with trypsin (before or after maleylation), chymotrypsin, pepsin, cyanogen bromide, or thermolysin yielded amino acid sequence information for all parts of the subunit and the primary structure of the whole protein chain was deduced (5S). It was found to contain 374 residues and is shown in Table I. An acetylated serine residue is at the N-terminus and the reactive cysteine residue is at position 46. Some residues are unevenly distributed (PS). Six of the seven histidine residues are in the N-terminal half of the molecule, the two tryptophan residues are in either terminal region, the four tyrosine residues are in the middle of the primary structure, and none of the 14 cysteine residues occur in the C-terminal quarter of the molecule. A characteristic distribution of hydrophobic residues was also noticed (93), which may now be partly correlated with the presence of large hydrophobic cores in the tertiary structure of the protein (Section II,C,3). Most regions of the primary structure were analyzed in many different overlapping peptides (92-9 ) with a corresponding increase in reliability. The structure is in excellent agreement with the total composition determined by acid hydrolysis (93). It is compatible with independently determined partial structures of... 

Thermolysin is a potent endopeptidase isolated from the organism Bacillus thermoproteolyticus. In contrast to the other zinc metalloenzymes discussed in this review, thermolysin has not yet been subjected to rigorous chemical and kinetic investigation. Therefore, although the complete amino acid sequence is known [184), and although Matthews and co-workers [43 6) have carried the X-ray structural analysis to 2.3 A resolution, it is neither possible to discuss the thermolysin catalytic mechanism nor to propose a role for zinc ion in thermolysin catalysis. Therefore, the following discussion is restricted to a brief review of the current status of the physical and chemical properties of thermolysin, and to a... 

A polypeptide containing 21 amino acids was hydrolyzed by thermolysin. The products of this treatment wctc Gly, lie, Val-Cys-So", Leu-Tyr-GIn, Val-Glu-Gln-Cys-Cys-Ala-Ser, and Leu-Glu-Asn-Tyr-Cys-Asn. When the same polypeptide was hydrolyzed by chymotrypsin, the products were Cys-Asn, Gln-Leu-Glu-Asn-Tyr, and Gly-Ile-Val-Glu-Gln-Cys-Cys-Ala-Ser-Val-Cys-Ser-Leu-Tyr. Give the amino acid sequence of this molecule. 

The complete amino-acid sequence was established by the use of two complementary degradative procedures, hydrolysis by trypsin and thermolysin. 

Chart VIII-5. The amino-acid sequence of clupeine YI with demonstration of peptides obtained by hydrolysis with carboxypeptidases B and A, trypsin and thermolysin [Ando and Suzuki, 1967 Suzuki and Ando, 1972 (2)]... 

The existence of two sets of overlapping peptides resulting from digestion with trypsin and thermolysin, combined with the results of the action of carboxypeptidases B and A used alternately, made it possible to deduce the total amino-acid sequence of clupeine YI as shown in Chart VIII-5. 

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)... 

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. 

To summarize for one component of salmine (S-AI) and three components of iridine (I-Ia, I-Ib and I-II) complete amino-acid sequences were deduced as shown in Chart VIII-9, from the information obtained from three sets of peptides resulting from digestion with trypsin, thermolysin, and Neutral Protease, as well as from the N- and C-terminal sequences. 

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. 

The protein contains only eight amino acids (C. L. Hew, personal communication). Alanine accounted for 60% of its total residues. The first N-terminal sequence of positions 1-28 was determined on a Beckman sequencer and confirmed by the isolation and analysis of overlapping thermolysin- and subtilisin-digested peptides. This sequence is ... 

Another example where aggregation of a library member drives its synthesis was recently reported by Ulijn ct al. [24, 25]. They used reversible amide bond formation, mediated by thermolysin, which is an enzyme that can catalyze both amide bond hydrolysis and formation, and is only moderately peptide-sequence-dependent. The authors reported that starting from dipeptides and fluorenyl-protected amino acids, the action of thermolysin gives rise to a dynamic mixture of peptides of different lengths (containing typically one to five amino acid residues). When using phenylalanine or leucine as the starting amino acids the... 

Pepsins and Pepsinogens.—The carbohydrate compositions and some sequences of amino-acids have been determined for glycopeptides derived from pepsinogens isolated from Japanese monkeys. Glycopeptides released by successive treatments of pepsinogen I with thermolysin and aminopeptidase contained 2-amino-... 

These figures show that thermolysin hydrolyzes no arginylarginine bonds at all, and furthermore, no bonds between basic amino acids (Hayashi, Iwai, and Ando, unpublished data). Thermolysin is thus an excellent new tool for sequence deter-... 

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