Human Red Cell Carbonic Anhydrase Research Articles

Kinetics and inhibition of carbonic anhydrase-catalyzed hydrolysis of 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone: Comparison with reactions of other substrates

The catalytic activities of human red cell carbonic anhydrase (EC 4.2.1.1) isozymes B and C for the hydrolysis of 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone have been compared with their activities towards three other substrates. The substrate specificity (measured as K cat/ K m) for either isozyme decreases in this order: CO 2 > 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone > acetaldehyde > p-nitrophenyl acetate. Unlike CO 2 hydration, enzyme B is slightly more active towards sultone hydrolysis than C. Despite these widely differing activities of both isozymes with regard to different substrates, the inhibition constants for anion and sulfonamide inhibitors are nearly independent of the substrate used. This suggests that the binding sites of these substrates in the enzyme are the same or nearly the same. Earlier studies on 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone from this and other laboratories had underestimated both the intrinsic activity and the susceptibility to anion inhibition of human carbonic anhydrase B. We now find that this was due to the use of acetonistrile as the substrate solvent, which is often contaminated with cyanide, a powerful inhibitor of carbonic anhydrase. The inhibition of human carbonic anhydrase B by several industrial batches of acetonitrile agrees completely with the spectrophotometrically determined cyanide content of these batches.

The carbon dioxide hydration activity of purified teleost red cell carbonic anhydrase. Inhibition by sulfonamides and anions

1. 1. Carbonic anhydrase is present as a single isozyme of molecular weight 29000 in the erythrocytes of the teleost, sheepshead ( Archosargus probatocephalus). 2. 2. The amino acid composition of this enzyme is generally similar to those of the mammalian isozymes of types I and II, although some notable incongruencies exist. 3. 3. The Michaelis-Menten kinetic constants ( K m and k cat) for CO 2 hydration by the sheepshead red cell carbonic anhydrase have been determined at pH 7.5–7.6 and at two temperatures, 1 and 25°C. This enzyme is not quite as active as the human red cell carbonic anhydrase isozyme II, although the turnover number is about 3 times that of human isozyme I at both temperatures. 4. 4. The pattern of inhibition of the sheepshead enzyme, by sulfonamides and monovalent anions, is similar to that of the mammalian high activity (type II) isozyme and is different from that exhibited by isozyme I.

Thermodynamics of carbonic anhydrase catalysis. A comparison between human isoenzymes B and C.

The CO2 hydration and HCO3- dehydration activities of human red cell carbonic anhydrase isozymes B and C (HCAB and HCAC) have been studied as a function of temperature from 0 degrees to 37 degrees C. The Arrhenius plots of ln kcat versus 1/T are linear for both isozymes in both hydration and dehydration reactions, indicating that the rate-determining steps remain unchanged over this temperature range. The 37 degrees C hydration kcat, at pH 7.5, is 13 X 10(5) s-1 for isozyme C and 0.71 X 10(5) s-1 for isozyme B. Km, for hydration, is 10 mM for C and 5 mM for B, and invariant with temperature. The uncatalyzed reactions are significantly affected by temperature, 30- to 40-fold rate enhancements being observed from 0 degrees to 37 degrees C. The enzyme-catalyzed processes are much less sensitive to temperature, the rate enhancements being 2- to 3-fold for HCAB and 5- to 6-fold for HCAC in this temperature range. These observations are consistent with a significant lowering of the free energy of activation by both isozymes. This effect is greater for C accounting for its higher catalytic power. The enthalpy of activation, at pH 7.5 and 8.2, in the rate-limiting step is considerably less for the B enzyme compared to C. This is, however, more than offset by a large negative entropy of activation in the case of HCAB. This observation indicates either a mechanistic difference in the rate-limiting events or a difference in the structural organizations of the active sites of the two isozymes, or both.

Current status of membrane-bound carbonic anhydrase.

We have studied the kinetic properties, and susceptibility to inhibition, of cytoplasmic and membrane carbonic anhydrase from dog kidneys, and attempted to place the data in the context of earlier work on this subject. The cytoplasmic enzyme thus far seems the same as human red cell carbonic anhydrase C, on the basis of kinetics, inhibition, amino acid composition and immunochemistry. On the other hand, the membrane enzyme is quite a different protein from either the cytoplasmic, or human red cell B or C. This enzyme is found in both luminal (brush border) and antiluminal (basolateral) fractions, and there appear no differences between the two. The turnover number (kcat) lies between those of B and C, and susceptibility to sulfonamide inhibition is two to 135-fold less than for the cytoplasmic enzyme, depending on the drug used. The usual difference is about fivefold. The KI for acetazolamide against the membrane enzyme is 10(-7) M, so that at the renal concentrations achieved at the usual in vivo doses (approximately 20 mg/kg) or used in current in situ perfusion work (both 10(-4) M) the enzyme is 99.9% inhibited. A striking difference between the membrane carbonic anhydrase and cytoplasmic or red cell B or C is its resistance to inhibition by halions. At 0.5 M chloride, there is no effect, whereas for the other three types inhibition ranges from 70%-99%. The membrane renal enzyme is also immunologically distinct from the other three types. The membrane enzyme has activity in its native state, but can be solubilized without loss of activity by treatment with Triton or sodium dodecyl sulfate. The actions of the renal carbonic anhydrases are depicted in a scheme that takes into account the protolysis of water, the attraction of H+ and of OH (and HCO3-) to the luminal and antiluminal membranes respectively, and the catalytic hydration and hydroxylation of CO2.

The nature of anion inhibition of human red cell carbonic anhydrases

We studied anionic inhibition of the reaction CO 2 + OH −⇄ HCO 3 − catalyzed by human red cell carbonic anhydrase B (I) and C (II), using iodide and cyanate. In the forward reaction with respect to CO 2 as the substrate, inhibition was mixed but favoring noncompetitive; the back reaction, with HCO 3 − as the substrate, yielded strict competitive kinetics. Mean inhibition constants, K I, in the pH range 7.2–7.5 are: iodide, 0.5 m m for enzyme B and 16 m m for C; cyanate, 0.8 μ m for B and 20 μ m for C. When OH − was considered as the substrate for the forward reaction, cyanate and chloride behaved as competitive inhibitors. The true inhibition constant ( K I 0) for cyanate (calculated for infinitely low OH −) is 0.4 μ m for enzyme B and 4 μ m for C. Apart from the difference in anion affinity and some 10-fold higher activity of C > B, the isozymes showed similar patterns of inhibition. Data agree with generally proposed mechanisms describing the active site as ZnH 2O with p K a of about 7.

A search for the function of human carbonic anhydrase B

Because of the very high activity and abundance of human red cell carbonic anhydrase C (carbamate hydrolase, EC 4.2.1.1), it seemed likely that the second isozyme, B, might not be essential for CO 2 metabolism. It was then found that physiological concentrations of Cl − inhibited catalysis of CO 2 hydration by the B enzyme (but not by type C), suggesting further that type B does not function in vivo as a carbonic anhydrase. The versatility of the catalytic activity of carbonic anhydrase for a number of ‘artificial’ substrates suggeested that enzyme B may be utilized in reactions of intermediary metabolism. A number of hydration, dehydration, decarboxylation, kinase, and phosphatase systems were tested to determine a possible physiological function for the enzyme. Results with eighteen possible substrates were negative and the possibility is discussed that mammalian carbonic anhydrase B is an evolutionary accident.

Inhibition by anions of human red cell carbonic anhydrase B: physiological and biochemical implications.

The hydration rate of CO2 catalyzed by human red cell carbonic anhydrase B is 92 percent reduced by the normal concentrations of chloride and bicarbonate in red cells. This reflects a general sensitivity of this reaction to halides and other anions, up to 87 times greater than the effect on red cell carbonic anhydrase C. The catalytic hydration of CO2 is generally more (up to 24 times) sensitive to inhibition by anions and sulfonamides than the dehydration of HCO3-, probably reflecting different mechanisms. The sensitivity of enzyme B to anion inhibition also depends upon the substrate, being much greater for CO2 than for certain esters. On the basis of the very low catalytic activity of B for CO2 in the presence of physiological concentration of chloride, and the fact that carbonic anhydrase C is effective for CO2 hydration (in the presence of chloride) at a rate 340 times greater than that of CO2 output from tissues, it appears that the biological role of enzyme B is not that of a carbonic anhydrase.

Response : Inhibition by Anions of Human Red Cell Carbonic Anhydrase B: Physiological and Biochemical Implications

<i>Response</i> : Inhibition by Anions of Human Red Cell Carbonic Anhydrase B: Physiological and Biochemical Implications

Inhibition by anions of human red cell carbonic anhydrase B: physiological and biochemical implications.

The hydration rate of CO2 catalyzed by human red cell carbonic anhydrase B is 92 percent reduced by the normal concentrations of chloride and bicarbonate in red cells. This reflects a general sensitivity of this reaction to halides and other anions, up to 87 times greater than the effect on red cell carbonic anhydrase C. The catalytic hydration of CO2 is generally more (up to 24 times) sensitive to inhibition by anions and sulfonamides than the dehydration of HCO3-, probably reflecting different mechanisms. The sensitivity of enzyme B to anion inhibition also depends upon the substrate, being much greater for CO2 than for certain esters. On the basis of the very low catalytic activity of B for CO2 in the presence of physiological concentration of chloride, and the fact that carbonic anhydrase C is effective for CO2 hydration (in the presence of chloride) at a rate 340 times greater than that of CO2 output from tissues, it appears that the biological role of enzyme B is not that of a carbonic anhydrase.

Characterization of a new variant of human red cell carbonic anhydrase I, CA If London (Glu-102 leads to Lys).

A new inherited variant of red cell carbonic anhydrase I (CA I), designated CA If London, was discovered during a survey of 1615 individuals from London, England. No electrophoretic variants of the other isozyme of carbonic anhydrase CA II, were observed in the same survey. Sequence analysis of a lysine-blocked tryptic peptide believed to contain the amino acid substitution in CA If showed that the glutamyl residue at position 102 had been substituted by a lysyl residue. This substitution results in a net increase of two positive charges in the mutant enzyme. Densitometric scanning of the electrophoretically separated forms in the variant hemolysate indicates that the levels of the normal and variant enzymes are approximately equal.

Substitution of lysine for threonine at position 100 in human carbonic anhydrase Id Michigan

The position of the amino acid substitution in the human red cell carbonic anhydrase I variant, CA Id Michigan, has been determined by sequence analysis of the altered tryptic peptide. The threonine to lysine substitution was found to be located at position 100, and is expressed as CA I 100 Lys.

Carbonic anhydrase, red cell membranes, and CO 2 transport.

Extracellular facilitation of CO2 transport by human red cell carbonic anhydrase has been demonstrated. Inhibition of this facilitation by DDT concentrated in the red cell membrane has also been shown, as well as the retention of a minor but significant amount of enzyme activity in washed bovine and human red cell ghosts. These findings are compatible with the concept that an appreciable catalytic effect on the hydration of extracellular CO2 is due to carbonic anhydrase associated with the cell membrane.

Effect of chlorthalidone binding on the electrophoretic properties of human red cell carbonic anhydrase isozymes.

The manner in which aromatic sulfonamide inhibitors bind to the carbonic anhydrases can provide us with useful information concerning the mechanisms of carbonic anhydrase action. Although considerable data are now available on carbonic anhydrase-sulfonamide binding kinetics (cf. Maren, 1967; Lindskog et al,, 1972), much less is known about structural changes in carbonic anhydrase brought about by sulfonamide binding. Recently, spectral analyses by King and Burgen (1970) and King and Roberts (1971) of sulfonamide complexes with human carbonic anhydrases, indicate that conformational changes occur in the enzymes as a result of the sulfonamide binding. In the present brief communication, we report some preliminary findings which may provide additional evidence for sulfonamide-induced conformational changes in carbonic anhydrase molecules.

The amino acid substitution and some chemical properties of a variant human erythrocyte carbonic anhydrase: carbonic anhydrase Id-Michigan.

Carbonic anhydrase IdMichigan, an electrophoretic variant of human red cell carbonic anhydrase I, was purified from erythrocytes obtained from an individual heterozygous for the trait. Primary structural analysis indicates that a lysine residue has exchanged for a threonine residue in the variant enzyme. After isolation, there was approximately 1.8 times as much normal as variant enzyme. Thermostability studies demonstrated that carbonic anhydrase Id was more thermolabile than the normal enzyme. The normal and variant enzymes showed no differences in specific carboxylesterase activity or CO2 hydratase activity. Utilizing the carboxylesterase activity toward β-naphthyl acetate, the normal and variant enzymes had similar Michaelis constants, pH profiles, and rates of inhibition by acetazolamide. Immunochemical studies did not demonstrate an antigenic difference for the variant enzyme.