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Tritium NMR spectroscopy has been used to examine the complexformed by [4-3H]benzenesulfon-amide and human carbonicanhydrase I. The results show that in solution the inhibitor forms a 1:1complex with the enzyme. A 100-spin computational model of the system,constructed with reference to crystallographic results, was used tointerpret tritium relaxation behavior and 3H{1H}NOEs. The analysis shows that the rate of dissociation of theenzyme–sulfonamide complex is 0.35 s−1 and thatthe aromatic ring of the inhibitor undergoes rapid rotation while complexed.

Fluorine NMR lineshape, relaxation and Overhauser effect data collected at 282 and 470 MHz have been used to obtain information about the nature of complexes formed between N-trifluoroacetyl-4-fluorophenylalanine and the enzyme chymotrypsin. Systems involving both enantiomers have been examined as well as derivatives of these in which the aromatic ring hydrogens have been replaced by deuterium. The enzyme-induced fluorine chemical shift effects and the dynamics of molecular motions of the fluorophenyl ring at the respective binding sites appear to be similar in both complexes and, where comparable, the results are in agreement with data obtained at lower frequencies that have been reported by other workers. The dynamics of the fluoroaromatic ring in these complexes are significantly different from those observed in a closely related acylated enzyme.

Powers and co-workers have provided evidence that thiobenzyl N-heptafluorobutyrylanthranilate (I) is an extremely potent inhibitor of serine proteases, especially alpha-chymotrypsin (Teshima, T., Griffin, J. C., and Powers, J. C. (1982) J. Biol. Chem. 257, 5085-5091). We have prepared additional derivatives of this structure in which fluorine substitutions have been made on the aromatic rings and have attempted to carry out fluorine NMR studies of the interaction of Powers' compound and these new derivatives with chymotrypsin. The solubility of all inhibitors examined in solvent systems compatible with the retention of native enzyme structure is extremely low. While some nmr evidence for complex formation could be obtained, preparations of the complexes examined were metastable and precipitation of the inhibitor eventually limits the amount of complex that can be present in solution to such low levels that nmr experiments are impractical. An unusual effect of solvent composition on fluorine chemical shifts suggests that the conformation of the inhibitors in aqueous solution and when bound to the enzyme is different from that in organic solvents.

Fluorine NMR has been used to examine complexes formed by 2-fluoro-, 3-fluoro-, and 2,5-difluorobenzenesulfonamide and human carbonic anhydrases I and II. The results show that all three inhibitors form complexes with both isozymes that have 2:1 inhibitor/enzyme stoichiometry. The fluorine spectra observed for all inhibitor-isozyme combinations are consistent either with rapid rotation of the aromatic ring of the inhibitor in the complexes or with preferential binding of only one of the two possible conformations of the inhibitors that are isomeric by virtue of rotation about the C1-C4 bond of the fluoro aromatic ring. Because ring rotation is slow in the case of the pentafluorobenzenesulfonamide-CA I complex, selective binding of rotamers is the explanation of these observations presently favored. A computer graphics study shows that formation of 2:1 complexes of CA I is feasible without appreciable distortion of the protein tertiary structure found in the crystalline state.

Binding of 4-fluorobenzenesulfonamide to human carbonic anhydrases I and II has been studied by proton, fluorine, and nitrogen-15 nuclear magnetic resonance spectroscopy. All three types of experiments provide evidence that the stoichiometry of the interaction of this inhibitor with both enzymes is 2 mol of inhibitor bound per mole of enzyme. Observations which suggest that the bound forms are involved in an exchange process that is rapid at room temperature but slower at 2 degrees C are described. Nitrogen-15 shift data show that the bound inhibitors are present at the active site as anions. The proton experiments indicate appreciable reorganization of the tertiary structure of the protein upon binding. Saturation-transfer experiments to determine the rate of dissociation of the inhibitor-enzyme complex lead to the conclusion that the dissociation process is more complicated than a simple free-bound equilibrium.

p-Trifluoromethylbenzenesulfonyl-α-chymotrypsin, an analog of tosylchymotrypsin, has been prepared and shown to be stable enough to permit fluorine nuclear magnetic resonance experiments. Up to four distinct trifluoromethyl resonances can be observed for the modified protein at 94.1 MHz even when the enzyme derivative is prepared from protein which has been purified by several methods. The resonances observed appear to represent proteins which are grossly similar as regards molecular size and the ability to bind and hydrolyze substrates, but nonetheless distinctive enough in the active-site region to produce appreciable chemical-shift effects.