The dehydration kinetics of human erythrocytic carbonic anhydrases B and C

Magid, Erik

doi:10.1016/0005-2744(68)90178-2

Cited by 35 publications

(12 citation statements)

References 16 publications

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“…Although these errors are rather large because of limitations inherent in the 'changing pH-indicator' method, it seems reasonable to conclude that an intramolecular proton transfer step is probably rate limiting in 2H20, while the catalytic rate may not be governed by a single step in ~H~O at 25°C (table 1). Perhaps this is a clue to the cause of the apparent discrepany with respect to the pH behaviour ofK d between our results [2] and those of Magid [13] who worked at 2°C. At temperatures above and below 25°C different steps may be rate limiting, and this could give rise to a variation of the shapes of the pH prof'des of the Michaelis-Menten parameters with temperature.…”

Section: Tentative Interpretationscontrasting

confidence: 49%

The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO₂ hydration and ester hydrolysis by HCO⁻₃

Steiner¹,

Jonsson²,

Lindskog³

1976

FEBS Letters

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Section: Tentative Interpretationscontrasting

confidence: 49%

The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO₂ hydration and ester hydrolysis by HCO⁻₃

Steiner¹,

Jonsson²,

Lindskog³

1976

FEBS Letters

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“…1, reaching a maximal value at acid pH with the same apparent pKa as the hydration reaction. The work of Magid (9) suggests that kcatHcOOS is independent of pH while KmHc°30 varies with pH. The experimental observation that the Michaelis-Menten parameters obey the Haldane relation of Eq.…”

Section: Steady-state Kinetics Of Carbonic Anhydrasementioning

confidence: 99%

The Catalytic Mechanism of Carbonic Anhydrase

Lindskog

Coleman

1973

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

315

184

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It is shown that an "inverse" relationship between the pH dependencies of the rates of hydration of CO2 and dehydration of HCO3-by carbonic anhydrase (EC 4.2.1.1) is a direct consequence of the thermodynamic equilibrium between CO2 and HCO3-and independent of any assumptions about the catalytic mechanism. It is further shown that proposed mechanisms for carbonic anhydrase involving HCO3-as the substrate in the dehydration reaction and a proton transfer reaction, EH+ E + H+, as an obligatory step during catalysis obey the rule of microscopic reversibility. This includes mechanisms in which the proton dissociation is from a zinc-coordinated water molecule. Such mechanisms can be in accord with the observed rapid turnover rates of the enzyme, since rapid proton exchange can occur with the buffer components, EH+ + B = E + BH+. Mechanisms in which H2CO3 is the substrate in dehydration avoid the proton-transfer step, but require that H2CO3 combines with enzyme more rapidly than in a diffusion-controlled reaction. Physicochemical evidence for and against a zinc-hydroxide mechanism is discussed.The zinc metalloenzyme carbonic anhydrase (EC 4.2.1.1) has been investigated by most techniques available for investigation of structure-function relationships (1-4). The crystal structure of the human C isoenzyme has been completed to a resolution of 2 A (2, 3). Yet there is little direct evidence identifying the catalytic and substrate binding groups in the enzyme. It is known that an ionizing group on the enzyme with a pKa near 7 is involved in catalysis, and that the titration of this group results in changes in the immediate environment of the metal ion. As to the identity of this group, the two major proposals are that its basic form represents (a) a zinc-coordinated hydroxide ion and (b) a basic amino-acid side chain, e.g., imidazole, directly or indirectly linked to the metal ion (1, 2, 4). Recently, Koenig and Brown (5) stated that proposed mechanisms involving a zinc-hydroxide as the active group violate the rule of microscopic reversibility, and they postulated that H2CO3 must be the appropriate substrate.As shown in the present paper, the zinc-hydroxide mechanism does not violate microscopic reversibility and, in fact, cannot be ruled out on kinetic or physicochemical grounds. Present data from physicochemical techniques in solution, chemical modification of the enzyme, and x-ray diffraction are analyzed as they pertain to the various proposals for the mechanism of action of carbonic anhydrase. where the equilibrium constants have the following values at 250 (6): Kh = 0.0026; K1 = 10-6 35 M; KHCO = 10-3"77 M.The reversible hydration of CO2 is a relatively slow reaction in the absence of a catalyst; the rate constant for hydration equals about 3.5 X 10-2 sec1.The enzyme-catalyzed reactionThe most active carbonic anhydrases, the human C and the bovine enzymes, catalyze both hydration of C02 and dehydration in the neutral pH range with rate constants between 105 and 106 sec'. This rapid turnover has posed some pr...

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“…The ionic strength of the assay mixture was kept constant and physiological by adding sodium sulphate. Sulphate was chosen because it has not been incriminated as catalyzing or inhibiting the reaction (Magid 1968, Khalifah 1971. Steady-state initial rates were obtained from the initial linear portions of the photographed reaction.…”

Section: Prepuration Of Purified Renal Soluble Carbonic Anhydrusementioning

confidence: 99%

Human renal cytoplasmic carbonic anhydrase Tissue levels and kinetic properties under near physiological conditions

Wistrand

1980

Acta Physiologica Scandinavica

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The cytoplasmic form of human renal carbonic anhydrase, CA-C (carbonate dehydratase, EC 4.2.1.1), purified by affinity chromatography, was characterized kinetically at 37 degrees C in 25 mM N-methyl imidazole buffer, I = 0.15, pH 7.1. using a pH-indicator stopped-flow technique. Under these conditions the rate constants for the uncatalyzed hydration of CO2 and dehydration of H2CO3 were 0.12 . s-1 and 0.60 . s-1, respectively. The kinetic parameters for CA-C were found to be: Hydration reaction, Km = 11.8 mM, V/[E]0 = 10.6 x 10(5) . s-1, dehydration reaction Km = 70 mM, V/[E]0 = 5 x 10(5) . s-1. In the hydration reaction CA-C was non-competitively inhibited by acetazolamide, Ki = 16 nM, sulfanilamide, Ki = 8 micrometer, and chlorothiazide, Ki = 1 micrometer. The levels of immunoassayable CA-C in cortex, medulla and papilla of perfused donor kidneys were 1.3, 1.0 and 0.6 mg enzyme protein/g tissue protein respectively which corresponded well with the levels measured catalytically. The erythrocyte form, HCA-B, was also detected immunochemically (approximately 0.1 mg/g protein) but is thought to be a contaminant. Calculations indicated that the uncatalyzed hydration of CO2 in the tubular cells can support 17 or 0.7% of the rate of urine acidification, dependent on whether the cellular alkaline pH-disequilibrium during acid secretion is 0.1 or 0.01 pH units, respectively. CA-C accelerates the hydration rate 6800-fold which enables the cell to sustain high rates of proton generation, while maintaining near CO2-equilibrium and maximal buffering capacity. Even at an assumed pH-disequilibrium of only 0.01 pH-unit, CA-C is present in 50-fold excess of apparent physiological needs. When the enzyme is inhibited the rate of the uncatalyzed reaction increases, which partly overcomes the effect of inhibition.

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The dehydration kinetics of human erythrocytic carbonic anhydrases B and C

Cited by 35 publications

References 16 publications

The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO₂ hydration and ester hydrolysis by HCO⁻₃

The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO₂ hydration and ester hydrolysis by HCO⁻₃

The Catalytic Mechanism of Carbonic Anhydrase

Human renal cytoplasmic carbonic anhydrase Tissue levels and kinetic properties under near physiological conditions

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The dehydration kinetics of human erythrocytic carbonic anhydrases B and C

Cited by 35 publications

References 16 publications

The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO2 hydration and ester hydrolysis by HCO−3

The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO2 hydration and ester hydrolysis by HCO−3

The Catalytic Mechanism of Carbonic Anhydrase

Human renal cytoplasmic carbonic anhydrase Tissue levels and kinetic properties under near physiological conditions

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Product

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The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO₂ hydration and ester hydrolysis by HCO⁻₃

The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO₂ hydration and ester hydrolysis by HCO⁻₃