A study of the histidine residues of human carbonic anhydrase B using 270 MHz proton magnetic resonance

Nine resonances in the 270 MHz proton magnetic resonance spectrum of human carbonic anhydrase B have been identified with imidazole C (2) protons of histidine residues, six of which are observed to titrate with p K a values in the range 4.7 to 7.4. The behaviour of the nine resonances has been studi...

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Veröffentlicht in:Journal of molecular biology 1974-12, Vol.90 (3), p.469-489
Hauptverfasser: Campbell, I.D., Lindskog, S., White, A.I.
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Sprache:eng
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Zusammenfassung:Nine resonances in the 270 MHz proton magnetic resonance spectrum of human carbonic anhydrase B have been identified with imidazole C (2) protons of histidine residues, six of which are observed to titrate with p K a values in the range 4.7 to 7.4. The behaviour of the nine resonances has been studied in the presence of the inhibitors, iodide, cyanide, acetate, hexacyanochromate, and imidazole. Measurements have also been made of the enzyme in its apo, cobalt, and mono-alkylated forms. Used in conjunction with the crystal structure, these results have enabled the tentative assignment of all nine resonances to particular histidine residues in the amino-acid sequence. Three of the active-site histidines at positions 64, 67, and 200 have low p K a values and cannot be directly linked to the activity of the enzyme. However, the resonances assigned to the three metal-liganding histidines do exhibit changes on anion binding and with pH, which parallel changes in the esterase activity. These results are consistent with the model of an ionizable water molecule bound to the zinc ion. Linewidth measurements of the resonances of the histidine residues on the enzyme surface are used to estimate pseudo-first-order rate constants of the order of 4 × 10 3 s −1 for D + exchange between imidazole N and solvent in the absence of buffer. These rates are observed to increase in the presence of small amounts of the buffers Tris and imidazole.
ISSN:0022-2836
1089-8638
DOI:10.1016/0022-2836(74)90229-0