Cooperative binding of two calcium ions to the double site of apothermolysin

Voordouw, Gerrit; Roche, Rodney S.

doi:10.1021/bi00721a024

Cited by 55 publications

(43 citation statements)

References 17 publications

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“…Experimentally, the quenching effect of Co(II) in the cobalt-Tb(III)-thermolysin relative to that of the zinc enzyme is F/Fo = 0.105 I 0.010. Since lanthanide ions most readily substitute for the Ca atom of site 1 of thermolysin (12,13), in the following it has been assumed that Tb(III) occupies this site, consistent with other considerations (see below). Co(II) occupies the Zn site (11) which is separated from the Tb(III) site by R = 13.7 A, based on x-ray structure analysis, yielding an Ro value of 19.6 A.…”

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confidence: 68%

“…Further, three of the four Ca sites may be occupied by trivalent lanthanide ions to the exclusion of all Ca atoms (12). The Ca site which is substituted most readily, number 1, is only 13.7 A from the active Zn binding site (12,13). The spectral overlap between the Tb(III) fluorescence and Co(II) absorption is sizable (Fig.…”

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confidence: 99%

“…In fact, using the value of Q = 0.51, n-4 = 0.32, K2 = and J = 5.92 X 10-16 M-l cm3, a value of Ro of 19.6 A is calculated which then yields a value of R of 13 Conformational changes affecting the tryptophan to Tb(III) energy transfer may account for both the small increase in fluorescence upon addition of Zn2+ to the zinc-free Tb(III)-thermolysin and the minor absorption spectral changes in the 290 nm region previously observed upon addition of zinc to apothermolysin (11).…”

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Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes.

Horrocks¹,

Holmquist²,

Vallée³

1975

Proc. Natl. Acad. Sci. U.S.A.

106

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The visible fluorescence of terbium(III) when bound to a calcium binding site of thermolysin is greatly enhanced with an excitation maximum at 280 nm but substitution of cobalt(II) for zinc at the-active site decreases the intensity by 89.5%. Treatment with N-bromosuccinimide quenches enzyme tryptophan and Tb(III) fluorescence to a similar extent and suggests the operation of tryptophan Tb(III) -Co(II) energy relay system in the enzyme. Dipoledipole radiationless energy transfer between the Tb(III) donor and the Co(II) acceptor can account for this quenching. The inherent characteristics of the metal pair limits the value of the orientation factor, K2, of the Fbrster equation, thereby reducing uncertainties in distance measurements by energy transfer compared with other systems. A quantum yield of 0.51 yields a value of Bo, the distance for 50% energy transfer, of 19.6 A, and a distance, R, between Tb(III) and Co(II) of 13.7 A, a value identical to that measured for the distance between the active site zinc atom and calcium atom number 1 by x-ray analysis in native thermolysin crystals.The limits of confidence of this measurement are discussed.Energy transfer between two different metal atom sites of a protein provides a new class of probes to measure intramolecular distances of biological macromolecules in solution.The substitution of paramagnetic, chromophoric metal ions, e.g., Co(II), Cu(II), or trivalent lanthanides, for spectroscopically silent ions which are native to metalloenzymes, e.g., Ca(II), Mg(II), or Zn(II), has proven increasingly useful in the study of their structure-function relationships (1). In the arsenal of spectroscopic techniques, fluorescence measurements offer particular advantages, since they can be employed in the study of biological molecules at relatively low concentrations. Certain of the lanthanide ions, principally Tb(III) and Eu(III), can exhibit considerably enhanced fluorescence when replacing Ca(II) in calcium-dependent proteins (2-4) or when bound to nucleic acids (5). Radiationless energy transfer is now an established means to probe distance relationships between organic fluorophoric donors and organic chromophoric acceptors (6, 7).We have now monitored energy transfer between metal ion donors and metal ion acceptors and thereby measured distances between different metal ion sites of thermolysin, a metalloendopeptidase whose metal content and three-dimensional structure are known (8,9 (Fig. 1). * To whom correspondence should be addressed.Hence, Co(II) and Tb(III) substituted thermolysin favors obh servation of inter-ion energy transfer and, owing to its known structure, provides a suitable benchmark for future distance determinations using this technique. EXPERIMENTAL Thermolysin, obtained from Calbiochem (San Diego, Calif.), was recrystallized and rendered zinc free as described (11). Enzyme concentrations were determined by measuring the absorbance at 280 nm, based on the reported absorbance E1%280 = 17.65 (14) and a molecular weight of 34,600 (8).The enzyme was...

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confidence: 68%

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confidence: 99%

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Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes.

Horrocks¹,

Holmquist²,

Vallée³

1975

Proc. Natl. Acad. Sci. U.S.A.

106

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“…Nine of the 13 residues implicated in the active site of thermolysin (4) (7). A common feature of both enzymes-is that they bind calcium, apparently necessary for stability (19)(20)(21)(22), and zinc, necessary for catalysis (23,24). It is therefore reasonable to inquire whether the metal ligands are mutable.…”

Section: Discussionmentioning

confidence: 99%

Evidence of homologous relationship between thermolysin and neutral protease A of Bacillus subtilis.

Levy¹,

Pangburn²,

Burstein³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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A comparison of the partial amino-acid sequence of neutral protease A from Bacillus subtilis with the structure of thermolysin (EC 3.4.24.4) from Bacillus thermoproteolyticus reveals that these two proteins are homologous. Of 171 residues placed in neutral protease (54% of the sequence), 83 residues (49%) occur in identical positions in thermolysin, and include nine of the 13 residues previously identified as components of the active site of thermolysin. This similarity provides support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. Since these enzymes differ markedly in their resistance to heat inactivation, a comparison of their structures may eventually provide a chemical basis for explaining the differences in their thermal stability. The metalloendopeptidases comprise a group of proteolytic enzymes that share a number of functional properties; these include a pH optimum near 7.0, inhibition by chelating agents-and resistance to inactivation by diisopropylphosphofluoridate or thiol reagents (1). Such "neutral proteases" have been isolated from a wide variety of sources that include bacteria, fungi, and more recently the brush border cells of rabbit kidney (1, 2). Apart from the notable exception of the amino-acid sequence (3) and three-dimensional structure (4) of thermolysin (EC 3.4.24.4), relatively little is known concerning the structural features of the metalloendopeptidases and their relation to function.Various studies have indicated that a neutral protease isolated from the culture medium of Bacilllus subtilis NRRLB3411 appears to be quite similar to thermolysin in amino-acid composition (5), kinetic behavior (6), and susceptibility to inhibitors (1, 7), which suggests that these two enzymes might be homologous. This investigation was undertaken to develop sequence data that would clarify the possible structural interrelationships between the neutral metalloendopeptidases, and in particular demonstrate that the neutral protease A from B. subtilis and the thermostable protease, thermolysin, are homologous proteins.

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“…comm. ); 106, Voordouw and Roche (1974);107, Robinson (1975);108, Hauert et al (1974); 109, Kay and Marsh (1959); I IO, Fukumoto et al (1967); 11 1, P. Horowitz (pers. comm.…”

Section: Cn Pace Et Almentioning

confidence: 99%

How to measure and predict the molar absorption coefficient of a protein

et al. 1995

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The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-3261 and is based on data from Edelhoch [ , Biochemistry 6:1948.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, These ~(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict E .

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Cooperative binding of two calcium ions to the double site of apothermolysin

Cited by 55 publications

References 17 publications

Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes.

Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes.

Evidence of homologous relationship between thermolysin and neutral protease A of Bacillus subtilis.

How to measure and predict the molar absorption coefficient of a protein

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