Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Limited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors (B values) determined crystallographically [Holmes, M.A., & Matthews, B.W. (1982) J. Mol. Biol. 160, 623-639]. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes.
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PMID:Correlation between sites of limited proteolysis and segmental mobility in thermolysin. 370 15

The mode of binding to thermolysin of the unsubstituted phosphoramidate inhibitor N-phosphoryl-L-leucinamide (P-Leu-NH2) has been determined crystallographically and refined at high resolution (R = 17.9% to 0.16-nm resolution). The mode of binding of the naturally occurring thermolysin inhibitor phosphoramidon reported previously [Weaver, L. H., Kester, W. R. and Matthews, B. W. (1977) J. Mol. Biol. 114, 119-132] has also been confirmed by crystallographic refinement (R = 17.4% to 0.23-nm resolution). Phosphoramidon binds to the enzyme with a single oxygen of the phosphoramidate moiety as a zinc ligand. Together with three ligands to the metal from the protein the resultant complex has approximately tetrahedral geometry. However, in the case of P-Leu-NH2, two of the phosphoramidate oxygens interact with the zinc to form a complex that tends towards pentacoordinate. In this respect, P-Leu-NH2 appears to be a better transition-state analog than is phosphoramidon. In addition, the phosphorus-nitrogen bond length in P-Leu-NH2 is 0.18 nm, suggesting that the nitrogen is protonated whereas the same bond in phosphoramidon is much shorter (0.15 nm) suggesting that the nitrogen does not carry a charge. In phosphoramidon the distance from the phosphoramide nitrogen to Glu-143 is 0.39 nm whereas in P-Leu-NH2 this distance decreases to 0.34 nm. Taken together, these observations provide additional evidence in support of the participation of pentacoordinate intermediates in the mechanism of action of thermolysin [Holmes, M. A. and Matthews, B. W. (1981) Biochemistry 20, 6912-6920] and the role of Glu-143 in first promoting the attack of a water molecule on the carbonyl carbon of the scissile bond and subsequently acting as a 'proton shuttle' to transfer the proton to the leaving nitrogen [Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5724-5729; Hangauer, D. G., Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5730-5741].
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PMID:Crystallographic structural analysis of phosphoramidates as inhibitors and transition-state analogs of thermolysin. 370 36

Storage proteins of the albumin solubility fraction from seeds of Bertholletia excelsa H.B.K. were separated by reversed-phase high-performance liquid chromatography and their primary structures were determined by gas-phase sequencing on intact polypeptides and on the overlapping tryptic and thermolysin peptides. The 2S storage proteins consist of two subunits linked by disulphide bridges. The large subunit (8.5 kDa) is expressed in at least six different isoforms while the small subunit (3.6 kDa) consists of only one form. These proteins are extremely rich in glutamine, glutamic acid, arginine and the sulphur-containing amino acids cysteine and methionine. One of the variants even contains a sequence of six methionine residues in a row. Comparison with known sequences of 2S proteins of other dicotyledonous plants shows limited but distinct sequence homology. In particular, the positions of the cysteine residues relative to each other appear to be completely conserved, suggesting that tertiary structure constraints imposed by disulphide bridges dominate sequence conservation. It has been proposed that the two subunits of a related protein (the Brassica napus storage protein) is cleaved from a precursor polypeptide [Crouch, M. L., Tenbarge, K. M., Simon, A. E. & Ferl, R. (1983) J. Mol. Appl. Genet. 2,273-283]. The amino acid sequence homology of the Brazil nut protein with the former suggests that a similar protein processing event could occur.
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PMID:The amino-acid sequence of the 2S sulphur-rich proteins from seeds of Brazil nut (Bertholletia excelsa H.B.K.). 375 80

The C-terminal fragment (Mr, 21,800) of colicin A (a bacterial toxin that kills sensitive Escherichia coli cells) has been crystallized. This fragment, which possesses the pore-forming activity of the toxin, resulted from thermolysin digestion of the entire molecule. The crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2) with a = b = 72.8 A, c = 170.4 A. They contain a dimer in the asymmetric unit and diffract to 2.7 A.
J Mol Biol 1986 Jul 05
PMID:Crystallization of the C-terminal domain of colicin A carrying the voltage-dependent pore activity of the protein. 378 97

A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50 degrees C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 50 degrees C, and those of lysozyme, RNase A and beta-lactoglobulin at pH 8 and 40 degrees C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.
J Mol Biol 1986 Aug 20
PMID:Unfolding rates of globular proteins determined by kinetics of proteolysis. 378 15

The COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin.
J Mol Biol 1985 Mar 20
PMID:Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure. 392 5

Among mutants of E. coli selected for temperature-sensitive growth, four were found to possess alterations in ribosomal proteins L7/L12. Of these, three apparently lack protein L7, the acetylated form of protein L12. Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E. coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min. Hence, the gene affected in these mutants was termed rimL. Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein. It was found that the assayed in vitro, in the high-speed supernatant prepared from mutant cells. These results indicated that the three mutants contain mutations in the gene rimL codes for an acetylating enzyme specific for ribosomal protein L12.
Mol Gen Genet 1981
PMID:Ribosomal protein modification in Escherichia coli. III. Studies of mutants lacking an acetylase activity specific for protein L12. 703 78

A set of 50 site-directed mutants of the Escherichia coli fms gene was constructed to delineate the residues of the active site of peptide deformylase, including the ligands of the zinc ion. In particular, because zinc is usually coordinate by Asp, Cys, Glu or His residues, all the corresponding codons were individually changed. The functional consequence of the substitutions was assessed by complementation of a fms-null strain with the help of vectors expressing the mutate genes. In addition to the mutations of the Cys90 codon, only those of the three conserved residues of the 132HEXXH136 motif of peptide deformylase prevented the indicator strain growing. Most enzyme variants were purified to homogeneity in a second step. Their characterization in vitro showed that the defects in complementation as observed in vivo corresponded to huge decreases of deformylation efficiency. The change of Glu88 also led to a significant decrease in catalytic rate. Unexpectedly, upon substitutions of Glu79 or of Glu83, the enzymes exhibited a strongly increased catalytic efficiency. The measurement of the content of zinc in each purified variant indicated that Cys90, His132 and His136 bound the metal ion. Zinc-free variants mutated at these positions were obtained and shown to display an increased sensitivity to proteolytic attack. Altogether, the data showed that both the presence of zinc and the conserved residues of the HEXXH motif were crucial for the activity of deformylase. This behaviour identified the enzyme as a member of the zinc metalloproteases superfamily. However, the unexpected participation in the binding of the zinc atom of Cys90, upstream from the HEXXH motif, suggested that peptide deformylase could be representative of a new sub-family, distinct from those of thermolysin and astacin.
J Mol Biol 1995 Nov 24
PMID:Mapping of the active site zinc ligands of peptide deformylase. 749 Jul 41

A computer procedure TFIT, which uses a molecular superposition force field to flexibly match test compounds to a 3D pharmacophore, was evaluated to find out whether it could reliably predict the bioactive conformations of flexible ligands. The program superposition force field optimizes the overlap of those atoms of the test ligand and template that are of similar chemical type, by applying an attractive force between atoms of the test ligand and template which are close together and of similar type (hydrogen bonding, charge, hydrophobicity). A procedure involving Monte Carlo torsion perturbations, followed by torsional energy minimization, is used to find conformations of the test ligand which cominimize the internal energy of the ligand and the superposition energy of ligand and template. The procedure was tested by applying it to a series of flexible ligands for which the bioactive conformation was known experimentally. The 15 molecules tested were inhibitors of thermolysin, HIV-1 protease or endothiapepsin for which X-ray structures of the bioactive conformation were available. For each enzyme, one of the molecules served as a template and the others, after being conformationally randomized, were fitted. The fitted conformation was then compared to the known binding geometry. The matching procedure was successful in predicting the bioactive conformations of many of the structures tested. Significant deviation from experimental results was found only for parts of molecules where it was readily apparent that the template did not contain sufficient information to accurately determine the bioactive conformation.
J Comput Aided Mol Des 1995 Jun
PMID:Flexible matching of test ligands to a 3D pharmacophore using a molecular superposition force field: comparison of predicted and experimental conformations of inhibitors of three enzymes. 756 76

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.
Plant Mol Biol 1995 Oct
PMID:The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts. 757 82


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