Amino acid sequence of thermolysin. Isolation and characterization of the fragments obtained by cleavage with cyanogen bromide

Titani, Koiti; Hermodson, Mark A.; Ericsson, Lowell H.; Walsh, Kenneth A.; Neurath, Hans

doi:10.1021/bi00763a007

Cited by 78 publications

(53 citation statements)

References 27 publications

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“…8-l for the partial specific volume. This figure compares well with the molecular mass (11.829 kDa) calculated from the known amino acid sequence of the fragment [29]. These results were confirmed by a single symmetrical sedimentation boundary obtained in high-speed sedimentation velocity experiments at 44000 rpm.…”

Section: Resultssupporting

confidence: 85%

“…Table 1 summarizes sedimentation coefficients and average molecular masses for thermolysin and its four C-terminal fragments investigated here. The calculated average molecular masses compare favourably with those calculated from the known amino acid sequences [29], indicating that the intact enzyme and all its fragments, except fragment 255-316 at with the tabulated molecular masses, assuming the protein fragment species represent hydrated spheres. Fragment 255 -316, at a working concentration of 0.6 mg/ml, gives a value for sZ0, which is more compatible with a dimer.…”

Section: Resultsmentioning

confidence: 51%

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Folding of thermolysin fragments

Vita

Fontana

Jaenicke

1989

European Journal of Biochemistry

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Sedimentation analysis in the analytical ultracentrifuge has been used to characterize the size and shape of thermolysin and a number of its fragments obtained by chemical or enzymatic cleavage of the protein. Four fragments (121 -316, 206-316, 225/226-316 and 255-316) originate from the C-terminal domain, and two (1 -155 and 1-205) from the N-terminal domain of the intact molecule. In aqueous solution at neutral pH the hydrodynamic properties of the C-terminal fragments, except 255 -316, are consistent with compact homogeneous monomers. Fragment 255-316 is a monomeric species below 0.08 mg/ml concentration and forms a dimer above this concentration. Dimerization does not lead to changes in fragment conformation, as determined by farultraviolet circular dichroic measurements, but to an increase of 5.6"C (to 68.2"C at 1.0 mg/ml) in the temperature for thermal unfolding and a corresponding increase of 4.6 kJ/mol in the free energy of unfolding. Fragments derived from the N-terminal domain show a strong tendency to form high-molecular-mass aggregates. Previous experiments utilizing circular dichroic measurements and antibody binding data suggested that the C-terminal fragments listed above are able to refold in aqueous solution at neutral pH into a stable conformation of native-, 331 -3401 (and references cited therein). Present data establish that all these C-terminal fragments are globular monomeric species in solution (at concentrations M 0.1 mg/ml) and thus represent 'isolated' domains (or subdomains) with intrinsic conformational stability typical of small globular proteins.There is clear evidence from X-ray analysis and limited proteolysis that the three-dimensional structure of monomeric globular proteins constituted by a polypeptide chain of more than about 100 amino acids results from the assembly of domains. Visual inspection of crystallographic models reveals the existence of compactly folded 'globules', corresponding to contiguous or non-contiguous segments of the polypeptide chain [I -41. Thus, protein domains (and subdomains) represent a specific level of organization ranking between the secondary structure elements (a-helix, strands) and the whole protein monomer in the hierarchical order of protein structure I5 -81. In a number of cases, protein fragments corresponding to protein domains have been shown to be folded into compact entities maintaining native-like characteristics, even in the absence of the residual polypeptide chain. The ability of fragments to fold autonomously yielding stable folding units suggests that domains play a role in the acquisition of the three-dimensional structure of globular proteins. Indeed, unfolding/refolding experiments clearly showed that domains represent intermediates on the sequential folding pathway finally leading to the native tertiary structure [9, 101. Previous To this aim, in this paper we report the hydrodynamic properties of four C-terminal and two N-terminal thermolysin fragments using sedimentation velocity and sedimentation equilibrium techniques. ...

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 51%

Folding of thermolysin fragments

Vita

Fontana

Jaenicke

1989

European Journal of Biochemistry

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“…Though there remains the possibility of polymorphism at residue 90, (i.e., Met or Asp occupying this position), we could obtain only one kind of cDNA clone synthesized by primer extension. The weakness of the Asp-Pro peptide bond to cyanogen bromide has been reported for thermolysin (Titani et al, 1972). Therefore, in sw-Achy, the amino acid at position 90 is thought to be always Asp, not Met.…”

Section: Discussionmentioning

confidence: 96%

Molecular cloning of silkworm (Bombyx mori) antichymotrypsin

Narumi

Hishida

Sasaki

et al. 1993

European Journal of Biochemistry

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The cDNA of silkworm (Bornbyx rnori) antichymotrypsin (sw-Achy) was cloned from larval fat body and its nucleotide sequence was determined. The deduced amino acid sequence of mature swAchy begins with Phel and ends with Phe384, with a preceding 16-amino-acid signal peptide. The amino-acid sequence similarities of sw-Achy with the serine-proteinase inhibitors (serpins) silkworm antitrypsin, tobacco hornworm alaserpin, human a-1-antitrypsin and human a-1 -antichymotrypsin were 29.6%, 30.3%, 26.1%, and 25.0%, respectively. The highly conserved amino acids in other serpins are also conserved in sw-Achy. sw-Achy is thought to be a new member of the serpin family. Multiple alignment of sw-Achy with 23 other kinds of serpin by the progressive method produced a phylogenetic tree in which all four insect serpins are grouped separately within one branch. The reactive site of sw-Achy with a-chymotrypsin was identified as Thr343-Ser344 by direct aminoacid sequence analysis of cleaved and purified protein.

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“…The tern (Pharmacia, Sweden). The purity was above 95% for all the samples amino acid sequence [2,3] and the three-dimensional (3D) strucexcept the tryptophan mutant~ The pl values of the three mutants, ture with several inhibitors [4,5] have been determined. Q119K, Q119R and Q119M, are 5.3, while that of other mutants and By analyzing the 3D structure, we were aware that the 119th wild type is 5.0.…”

Section: Mutants Of Thermolysinmentioning

confidence: 99%

Remarkable activity enhancement of thermolysin mutants

et al. 1995

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Most attempts to modify the properties of enzymes by effective in producing the electrostatic field at an activesite of amino acid substitution around the active sites have resulted in the enzyme, the 143rd glutamate, judging from the electrostatic suppression of the biological activity, suggesting that the structure of natural enzymes should be almost optimized evolutionally calculation, indicating that the change of electric charge at this to show the highest activity. In contrast, we found an interesting site may affect the activity [6]. Secondly, the 119th glutaminesite of a well-known metalloendopeptidase, thermolysin (EC.was estimated to play a role in stabilizing the structureby form-3.4.24.4), where almost all the amino acid replacement causes a ing a hydrogen bond with the hydroxyl group of the 103rd remarkable increase in the hydrolytic activity. Negative correlaserine, as already proposed for the neutral protease from Baciltion between the activity and the thermal stability was observed, lus stearothermophilus [7]. The breakage of the hydrogen bond The flexibility around the substrate binding site is suggested to may affect the activity and the thermal stability with changing be a key to the correlation. Nature may have selected the amino the flexibility around the substrate binding site. acid at this site, which suppresses the flexibility of the molecule, It should be noted that the cDNA sequencing [8] and our to get the highest thermal stability at the expense of the activity, re-examination of direct amino acid sequencing of the enzyme Key words: Enzyme stability; Activity; Design principle; [9] have corrected the known sequence by two residues, and Amino acid mutation that the l l9th happens to correspond to one of them. The corrected sequence is identical with that of a neutral protease from Bacillus stearothermophilus [7].

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Amino acid sequence of thermolysin. Isolation and characterization of the fragments obtained by cleavage with cyanogen bromide

Cited by 78 publications

References 27 publications

Folding of thermolysin fragments

Folding of thermolysin fragments

Molecular cloning of silkworm (Bombyx mori) antichymotrypsin

Remarkable activity enhancement of thermolysin mutants

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