Hydrogen Ion Equilibria and the Chemical Modification of Lysine and Tyrosine Residues in Bovine Carbonic Anhydrase B

Abstract

1. Potentiometric titrations of bovine carbonic anhydrase B have been performed. On titration with acid from the isoionic point, pH 5.65, seven histidines, which are masked in the native enzyme, become protonated during a conformational change occurring in a narrow pH-range near pH 4. Concomitantly, the enzyme activity is lost. On back-titration of the acid-denatured enzyme from pH 2, all 11 histidines are titratable. The denatured enzyme shows an increase of titratable groups in the alkaline pH-range as well.2. Spectrophotometric titrations a t alkaline p H show that most of the tyrosines are masked in the native enzyme. Only one tyrosine seems to titrate freely but with a rather high value of the intrinsic pK (10.8). The remaining seven tyrosines ionize above pH 12 during a time-dependent change leading to activity loss.3. Tetranitromethane reacts with only one tyrosine in the native enzyme. The modified enzyme retains full catalytic activity. 4.Methyl acetimidate reacts with a t least 95 of the lysines, including the carboxyl-terminal residue, without loss of activity. The number of titratable groups in the pH region 9 to I1 is accordingly decreased. Several forms of carbonic anhydrase have been isolated from human and bovine erythrocytes [l-41.These enzyme forms have quite similar properties, but marked differences in details have been found, including differences in catalytic behavior [2,5]. All forms contain one zinc ion which is essential for activity [6 -81, and they have about the same moleccular weight [3,9,10]. The amino acid compositions of the human forms B and C and the bovine form B, are considerably different [3,9,11], but it has recently been shown that there are some sequences common for two or all three forms [12]. The results of measurements of optical rotatory dispersion [13-161 and circular dichroism [I31 indicate common features but also distinct differences in the conformations of the various enzyme forms.We have continued the comparative approach to the problem of the structure-activity relationship in carbonic anhydrase and present in this paper the results of studies on the hydrogen ion equilibria of the bovine enzyme, form B. We have found that the titration behavior of this form differs from those of the human forms B and C [17,18] as expected from the differences in amino acid compositions and, in addition, that a larger number of tyrosyl residues seem to be masked in the bovine enzyme. Most of the histidyl residues are masked in the native states of Enzyme. Carbonic anhydrase or carbonate hydro-lyase (EC 4.2.1.1).both the human and bovine forms and become protonized only after denaturation in acid. MATERIALS AND METHODS Bovine Erythrocyte Curbonic Anhydruse BThis enzyme was prepared as previously described by Lindskog [l]. The final zone electrophoresis step was omitted, however, and replaced by rechromatography on a DEAE-cellulose column (d, 4 c m ; 1, 40 cm). This procedure gave a higher yield of form B than the original method, and the enzyme had the same degree of purity as jud...

An essential tyrosine residue of Aspergillus polygalacturonase

“…The change in absorptivity at 295 nm associated with ionization of one tyrosine residue was calculated from the relation: 2540 M "! xcm -t [17], where M is the molecular mass of the enzyme.…”

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…Nilsson and Lindskog demonstrated that, near pH 4, the expansion of BCA II occurs simultaneously with the protonation of seven buried His residues. 753 Because the Zn II cofactor is coordinated by three His residues, it is not surprising there is no measurable enzymatic activity at pH 3.7.…”

Studies of the Esterase Activity and the Anion Inhibition of Bovine Zinc and Cobalt Carbonic Anhydrases

“…Bovine erythrocyte carbonic anhydrase B was prepared according to the method of Lindskog [13] except that the final zone electrophoresis step was replaced by rechromatography on DEAE-cellulose [14]. Procedures spectrophotometrically at 280 mp taking the molecular extinction coefficient e2*/, = 57,000 M-l x cm-l and a molecular weight of 30,000 [17].…”