RESUMEN
The present work demonstrates that the high-activity zinc metalloenzyme, carbonic anhydrase (CA II) from bovine erythrocytes is inhibited by the cyclic sulfimide, saccharin, and 2- and 4-carbobenzoxybenzene sulfonamide. A spectrophotometric method was employed to monitor the enzymatically catalyzed hydrolysis of p-nitrophenyl acetate by following the increase in absorbance at 410 nm which accompanies p-nitrophenoxide/p-nitrophenol formation. The more rapid enzymatic hydration of CO2 was monitored by using a stopped-flow spectrophotometer as well as by a modified colorimetric method of Wilbur and Anderson. The studies show that, at a given molar ratio of inhibitor to enzyme, the degree of inhibition of the enzymaic hydration of CO2 and hydrolysis of p-nitrophenyl acetate by the inhibitory compounds is essentially the same. Kinetic analyses were made at 25.0 degrees at pH 6.5 (MES buffers), pH 6.9 (HEPES buffers) and pH 7.9 (HEPES buffers) with ionic strength regulated by the addition of appropriate quantities of sodium sulfate. Lineweaver-Burk plots were used to evaluate apparent inhibition constants for each of the three inhibitors. For all the inhibitors studied, inhibition appears to be mixed (competitive/noncompetitive). For saccharin in the presence of sodium sulfate, the extent of inhibition is considerably decreased. It was found for the three inhibitors that the inhibitory potency decreases with increasing pH, and that the inhibitory potency is extremely sensitive to the shape of these rather closely related molecules. For example, apparent inhibition constants for the enzymatic hydrolysis of p-nitrophenyl acetate at pH 6.9 were Ki (saccharin) = 0.20 mM, Ki (2-carbobenzoxybenzene sulfonamide) = 0.54 mM and Ki (4-carbobenzoxybenzene sulfonamide) = 1.6 microM. For the enzymatic hydration of CO2 at pH 6.9, 0.10 mM saccharin caused 50% inhibition while 7.0 nM 4-carbobenzoxybenzene sulfonamide resulted in 50% inhibition. The results suggest that sulfonamide inhibition is caused by formation of a monodentate ligand at the zinc ion of the enzyme active site and that the more linear 4-carbobenzoxybenzene sulfonamide is better able to enter a conical enzyme active site than is 2-carbobenzoxybenzene sulfonamide or saccharin.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Sacarina/farmacología , Sulfonamidas/farmacología , Algoritmos , Animales , Tampones (Química) , Dióxido de Carbono/metabolismo , Bovinos , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
Xanthine oxidase (XO), catalyzes the sequential oxidation of hypoxanthine to xanthine and then to uric acid. The enzyme also catalyzes the oxidation of aldehydes to their corresponding carboxylic acids. In the present work we investigate the extent of inhibition of the xanthine oxidase-catalyzed oxidation of hypoxanthine by acetaldehyde/acetaldehyde hydrate system. At room temperature, aqueous solutions of acetaldehyde exist as equilibrated mixtures containing similar quantities of the aldehyde, CH3CHO and its hydrate CH3CH(OH)2. To determine whether acetaldehyde or its hydrate interacts with the enzyme to cause inhibition, the time course of enzymatic inhibition was observed in deoxygenated solutions of xanthine oxidase initially incubated with neat acetaldehyde and compared to that in which the enzyme was initially incubated with aqueous solutions containing both the aldehyde and its hydrate. Our results show that unhydrated acetaldehyde inhibits XO and that the inhibition of the XO-catalyzed oxidation of hypoxanthine progressively increases as the aldehyde is incubated with the enzyme. The data, taken together, suggest that enzymatic inhibition is the result of the reversible formation of covalently bound XO-acetaldehyde inhibitory compound. This investigation also demonstrates that the enzymatic oxidations of hypoxanthine and acetaldehyde take place on the same active site in XO.
Asunto(s)
Acetaldehído/farmacología , Xantina Oxidasa/antagonistas & inhibidores , Hipoxantina/metabolismo , Cinética , Soluciones , AguaRESUMEN
The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.