Drug-protein interactions. Refined structures of three sulfonamide drug complexes of human carbonic anhydrase I enzyme.

N-unsubstituted sulfonamide drugs are widely used for opthalmic disorders. Inhibition of carbonic anhydrase enzyme is believed to be the chief reason for their therapeutic effects. Structures of three such sulfonamide drugs complexed to human carbonic anhydrase I enzyme (HCAI) refined crystallographically at 2 A resolution are reported here. The drug molecules are all bound in the active site of the enzyme, but among themselves show differences in the orientations of the sulfamido groups interacting with the essential zinc ion in the active site. The activity linked solvent molecule coordinated to zinc in the native enzyme is displaced by all the three sulfonamides. The active site loop of Leu198, Thr199 and His200 has been identified to be important for binding of the drug molecules due to their appreciable atomic displacements and intra-molecular hydrogen bonds arising out of their interactions with the sulfonamides. These interactions along with active site charge requirements are proposed to be responsible for the orientational differences of the sulfamido groups and also for differences in the inhibitory powers of the drugs. A hydrogen bond network involving solvent molecules and active site residues His200 and His67 amongst others in the native enzyme, is disrupted upon binding of methazolamide but not in the other two sulfonamides. This is the first crystallographic evidence of the possible involvement of His200 in the inhibition of HCAI. An important role of Thr199 in distinguishing between the substrate and inhibitor binding modes of HCO3- to the enzyme at high pH is also inferred.

[Study of the binding of diuretics by serum proteins according to changes in tryptophan fluorescence]. / Izuchenie sviazyvaniia diuretikov s belkami syvorotki krovi po izmeneniiu triptofanovoi fliuorestsentsii.

The effect of diuretics on tryptophan fluorescence of blood serum proteins divided by electrophoresis was studied. It was shown that ethacrynic acid produces the most significant extinction of tryptophan fluorescence in albumin fraction and novurit in globulin fraction. Furosemide possessing a high affinity for all three obtained fractions of protein does not exhibit a preferential binding to one or another of these fractions. It was also found that furosemide and ethacrynic acid by binding to human serum albumin molecule produce its conformational alterations. Mercury diuretic does not possess such effect.

Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: a pentacoordinated binding of the SCN- ion to the zinc at high pH.

The binding of four inhibitors--mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion--to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography. The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 A resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206. The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 A, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closest to the zinc ion is 3.1 A away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc. The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 A resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 A from the zinc ion but shifted 1.3 A with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the O gamma l atom of Thr-199. This is due to the inability of the O gamma l atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called "deep" water molecule of the native enzyme. The zinc-bound water molecule is 2.2 A from the zinc ion and 2.4 A from the SCN- nitrogen. In addition, this water is hydrogen bonded to the O gamma l atom of Thr-199 and to another water molecule. We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.

[Diuretic binding with biological membranes studied by means of a fluorescent probe]. / Izuchenie sviazyvaniia diuretikov s biologicheskimi membranami metodom fliuorestsentnogo zonda.

The binding of furosemide, novurit and ethacrynic acid with human serum albumin, phospholipid liposomes, mitochondria and plasmatic membranes of rat kidneys was studied by the fluorescent probe 1-anilino naphthalene-8-sulfonate (1,8-ANS) method. It is shown that ethacrynic acid has high associative constants with serum albumin molecule and plasmatic membranes. Novurit has the greatest affinity with rat kidney mitochondria. Furosemide does not bind with plasmatic membranes and has very low constants of association with mitochondria and phospholipid liposomes.

[Mechanism of action of diuretics studied by a fluorescent probe method]. / Izuchenie mekhanizma deistviia diuretikov metodom fliuorestsentnogo zonda.

The mercuric-xanthine diuretic novurit, ethacrynic acid and furosemide reduced the fluorescence of 1,8-anilinonaphthalene-8-sulfonate (1,8-ANS) in a suspension of phospholipid liposomes and human serum albumin solution. Ethacrynic acid and furosemide eliminated the fluorescence of 1,8-ANS in a suspension of non-energized mitochondria of rat kidney, while novurit provoked fluorescence intensification. The constants of novurit and ethacrynic acid binding with kidney mitochondria determined by the degree of changes in 1,8-ANS fluorescence variation appeared significantly higher than the constants of binding with phospholipids and serum albumin. However furosemide did not show a predominant binding with kidney mitochondria.

Screening of common bacteria capable of demethylation of methylmercuric chloride.

In summary, 21 bacteria were capable or degrading methylmercury chloride as measured by the disappearance of that species from the soil culture media. Disappearance of the methylated form was also accompanied by loss of total mercury in the culture media--probably as a result of reduction of the initial metabolite to volatile Hgo. The role of bacteria in the reduction step is not clear at this time although several authors (SUMMERS et al. 1972 and 1973; BISOGNI and LAWRENCE 1973 ) have suggested facilitated reduction rates in the presence of microorganisms.