Carbon-13 nuclear magnetic resonance as a probe of side chain orientation and mobility in carboxymethylated human carbonic anhydrase B

Schoot Uiterkamp, A.J.; Armitage, I.M.; Prestegard, J.H.; Slomski, J.; Coleman, J.E.

Biochemistry 17(18): 3730-3736

1978


ISSN/ISBN: 0006-2960
PMID: 100136
DOI: 10.1021/bi00611a009
Accession: 068520714

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Abstract
13C NMR spectra of [1-13C]- and [2-13C]carboxymethyl His-200 human carbonic anhydrase B [EC 4.2.1.1] were obtained as a function of pH and in the presence and absence of the active site Zn(II) or Cd(II) ion. Chemical shifts of the 1-13C show that the carboxyl is sensitive to 2 ionization processes, with apparent pKa of 7.2 and 9.9, respectively. These are assigned to the deprotonation of the N.pi. of His-200 and the breaking of the coordination bond between the carboxyl oxygen and the Zn(II) ion, respectively. Assignment of the lower pKa to that of the N.pi. is supported by the observation of this same ionization in the chemical shift of the 2-13C resonance showing the signal from the methylene carbon to undergo the same upfield shift as is observed on the ionization of the N.pi. in N.tau.-carboxymethylated histidine. The high pH ionization process is not reflected in the resonance of the methylene carbon. No changes in the chemical shifts vs. pH are observed for both the [1-13C]- and [2-13C]carboxymethyl apocarbonic anhydrase, suggesting that the pKa of the N.pi. has shifted at least 1 pH unit to acid pH, and must reflect significant conformational changes in the active center. Cd(II) carboxymethyl carbonic anhydrase shows 13C chemical shifts identical with those of the apoenzyme. Since the Cd(II) at the active site is known to bond external donor groups very weakly, the data suggest that the changes in conformation are related to Zn(II)-carboxylate coordination. Changes in the mobility of the carboxymethyl group were assessed by a relaxation analysis which relates the relative line widths of the central and outer lines of the [13C]methylene triplet to the internal rotational motion of the group relative to the protein. At neutral pH this group no internal motion, compatible with its coordination to the active site metal ion. At pH 10.6 significant internal motion is present, compatible with breaking of the coordination bond in competition with -OH binding.