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Mammalian arginases I and II require an intact binuclear manganese cluster for the hydrolysis of l-arginine to generate l-ornithine and urea. Although arginase isozymes differ in terms of their tissue distribution, cellular localization, and metabolic function, each employs a metal-activated hydroxide mechanism for catalysis. To date, the best arginase inhibitors are those bearing N-hydroxyguanidinium or boronic acid “warheads” that can bridge the binuclear manganese cluster. Strikingly, the trigonal planar boronic acids undergo nucleophilic attack by hydroxide ion to form tetrahedral boronate anions that mimic the tetrahedral intermediate and its flanking transition states in the arginase mechanism. Given their affinity and specificity for arginase, boronic acid inhibitors are especially useful for probing the role of arginase in living systems. Arginase can regulate l-arginine bioavailability to nitric oxide synthase by depleting the substrate pool for NO biosynthesis, so arginase inhibition can enhance the substrate pool for NO biosynthesis. Accordingly, arginase inhibition can enhance NO-dependent physiological processes, such as the smooth muscle relaxation required for sexual arousal:  administration of arginase inhibitors in vitro and in vivo enhances erectile function and engorgement in the male and female genitalia. Therefore, arginase is a potential therapeutic target for the treatment of sexual arousal disorders in men and women.

Carbonic anhydrase inhibitors are effective in lowering intraocular pressure, the primary indication of glaucoma. Human carbonic anhydrase II, and possibly carbonic anhydrase IV (CAII and CAIV, respectively), help regulate fluid secretion into the anterior chamber of the eye. Because inhibitors currently formulated as drugs to treat glaucoma were designed to target CAII, an understanding of the structural basis of CAII−CAIV discrimination by inhibitors would be useful for probing the role of each isozyme in the etiology of the disease. Here, we report the X-ray crystal structures of three novel thieno[3,2-e]-1,2-thiazine-6-sulfonamides complexed with CAII and the computationally predicted structures of the same compounds complexed with CAIV. All three compounds bind with similar affinity to CAII, but they bind with up to 100-fold lower affinities to CAIV. Comparisons of experimentally determined structures of CAII−inhibitor complexes and computationally predicted structures of CAIV−inhibitor complexes allow us to rationalize these affinity trends and outline molecular features that may contribute to high-affinity inhibitor binding to CAIV. This study demonstrates how experimental structure determination methods and computational structure prediction methods can be used together to answer questions that cannot be answered by either method alone.

The structure of the trimeric, manganese metalloenzyme, rat liver arginase, has been previously determined at 2.1-A resolution (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W., (1996) Nature 383, 554-557). A key feature of this structure is a novel S-shaped oligomerization motif at the carboxyl terminus of the protein that mediates approximately 54% of the intermonomer contacts. Arg-308, located within this oligomerization motif, nucleates a series of intramonomer and intermonomer salt links. In contrast to the trimeric wild-type enzyme, the R308A, R308E, and R308K variants of arginase exist as monomeric species, as determined by gel filtration and analytical ultracentrifugation, indicating that mutation of Arg-308 shifts the equilibrium for trimer dissociation by at least a factor of 10(5). These monomeric arginase variants are catalytically active, with k(cat)/K(m) values that are 13-17% of the value for wild-type enzyme. The arginase variants are characterized by decreased temperature stability relative to the wild-type enzyme. Differential scanning calorimetry shows that the midpoint temperature for unfolding of the Arg-308 variants is in the range of 63.6-65.5 degrees C, while the corresponding value for the wild-type enzyme is 70 degrees C. The three-dimensional structure of the R308K variant has been determined at 3-A resolution. At the high protein concentrations utilized in the crystallizations, this variant exists as a trimer, but weakened salt link interactions are observed for Lys-308.

Linear free energy relationships between binding affinity and hydrophobicity for a library of fluoroaromatic inhibitors of F131V carbonic anhydrase II (CA) implicate three modes of interaction. X-ray crystal structures suggest that F131 interacts with fluoroaromatic inhibitors, while P202, on the opposite side of the active site cleft, serves as the site of the hydrophobic contact in the case of the F131V mutant. 2-Fluorinated compounds bind more tightly, perhaps due to the field effect of the nearby fluorine on the acidity of the amide proton.

The structure of the complex between carboxypeptidase A alpha (EC 3.4.17.1) and the ketonic substrate analogue 5-benzamido-2-benzyl-4-oxopentanoic acid (BOP) has been determined by x-ray crystallographic methods to a resolution of 1.7 A (final R = 0.191). Interestingly, BOP was observed to bind to the active site of carboxypeptidase A alpha as the covalent hydrate adduct. Because BOP is probably less than 0.2% hydrated in aqueous solution, this result was unexpected. One possibility is that the zinc-bound water of the native enzyme added to the ketone carbonyl. Alternatively, the enzyme may preferentially scavenge the hydrated ketone as it is continuously maintained at equilibrium in the solution in which the carboxypeptidase A alpha crystals were immersed. In either case, this mode of binding of BOP to carboxypeptidase A alpha provides an example of the preferred binding of a model of a structure along the reaction coordinate of a hydrolytic reaction.

The complex of carboxypeptidase A (CPA) with 5-amino-(N-t-butoxycarbonyl)-2-benzyl-4-oxo-6-phenylhexanoic acid (BBP), the ketomethylene substrate analogue of the peptide substrate N-(t-butoxycarbonyl)-L-phenylalanyl-L-phenylalanine, was studied by x-ray crystallographic methods. Interestingly, the enzyme specifically binds only one of four stereoisomers of BBP that were present in the buffer solution in which the CPA crystals were soaked. Furthermore, the species observed to bind to the enzyme is the hydrated form of the ketone. This is rather surprising since the hydrated form of BBP is expected to be present in aqueous solution at a concentration of less than 0.2%. Hence, the enzyme-inhibitor complex is most stable with a species resembling a structure along the reaction coordinate of a chemical reaction rather than a species resembling a reactant or a product. Important structural information regarding the catalytic conformations of active-site residues spanning the S'1-S2 subsites of the enzyme is provided from the results of these x-ray diffraction experiments. The structure of the CPA-hydrated BBP complex provides support for a promoted-water hydrolytic mechanism, although it is not certain whether the enzyme has actually participated in the hydration reaction at the ketone carbonyl of BBP.

The three-dimensional structure of human carbonic anhydrase II (CAII) complexed with the sulfonamide fluorophore 5-dimethylamino-1-naphthalene sulfonamide (dansylamide) has been determined to 2.1-A resolution by x-ray crystallographic methods. Unlike other arylsulfonamide inhibitors of CAII, the naphthyl ring of dansylamide binds in a hydrophobic pocket in the active site, making van der Waals contacts with Val-121, Phe-131, Val-143, Leu-198, and Trp-209. Interestingly, a conformational change of Leu-198 is required to accommodate dansylamide binding, which rationalizes the enhanced dansylamide affinity measured for certain Leu-198 variants (Nair, S. K., Krebs, J.F., Christianson, D. W., and Fierke, C. A. (1995) Biochemistry 34, 3981-3989). Modeling studies indicate that a second binding mode, in which the fused aromatic ring is rotated out of the hydrophobic pocket, is sterically feasible. Both experimentally observed and modeled binding modes have implications for new leads in the design of avid CAII inhibitors. Finally, the structure of the CAII-dansylamide complex has implications for its exploitation in zinc biosensor applications, and possible routes toward the optimization of fluorophore design are considered on the basis on this structure.

The mode of binding of the competitive inhibitor 2-benzyl-3-formylpropanoic acid to the active site of carboxypeptidase A has been studied by x-ray diffraction methods to a resolution of 1.7 A. The actual species bound to the enzyme was determined to be the gem-diol resulting from covalent hydration at the aldehyde carbonyl. Details relating to the process of association of inhibitor with enzyme are unknown at this time: the free aldehyde could initially bind to the enzyme and subsequently undergo catalytic hydration; or, the hydrate itself could be the species initially binding to the enzyme, because it does exist to a high degree (25%) in aqueous solution. Nevertheless, the structure of the complex reported is reminiscent of a possible tetrahedral intermediate that would be encountered in a general base hydrolytic mechanism. Of course, other mechanistic proposals, such as the anhydride pathway, cannot be ruled out simply on the basis of the structure of this enzyme-inhibitor complex.

A high-resolution x-ray crystallographic investigation of the complex between carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the slowly hydrolyzed substrate glycyl-L-tyrosine was done at -9 degrees C. Although this enzyme-substrate complex has been the subject of earlier crystallographic investigation, a higher resolution electron-density map of the complex with greater occupancy of the substrate was desired. All crystal chemistry (i.e., crystal soaking and x-ray data collection) was performed on a diffractometer-mounted flow cell, in which the crystal was immobilized. The x-ray data to 1.6-A resolution have yielded a well-resolved structure in which the zinc ion of the active site is five-coordinate: three enzyme residues (glutamate-72, histidine-69, and histidine-196) and the carbonyl oxygen and amino terminus of glycyl-L-tyrosine complete the coordination polyhedron of the metal. These results confirm that this substrate may be bound in a nonproductive manner, because the hydrolytically important zinc-bound water has been displaced and excluded from the active site. It is likely that all dipeptide substrates of carboxypeptidase A that carry an unprotected amino terminus are poor substrates because of such favorable bidentate coordination to the metal ion of the active site.

The structures of the complexes of carboxypeptidase A with the amino acids D-phenylalanine and D-tyrosine are reported as determined by x-ray crystallographic methods to a resolution of 2.0 Å. In each individual study one molecule of amino acids binds to the enzyme in the COOH-terminal hydrophobic pocket: the carboxylate of the bound ligand salt links with Arg-145, and the α-amino group salt links with Glu-270. The carboxylate of Glu-270 must break its hydrogen bond with the native zinc-bound water molecule in order to exploit the latter interaction. This result is in accord with spectroscopic studies which indicate that the binding of D or L amino acids (or analogues thereof) allows for more facile displacement of the metal-bound water by anions (Bicknell, R., Schaffer, A., Bertini, I., Luchinat, C., Vallee, B. L., and Auld, D. S. (1988) Biochemistry27, 1050-1057). Additionally, we observe a significant movement of the zinc-bound water molecule (∼ 1 A) upon the binding of D-ligands. We propose that this unanticipated movement also contributes to anion sensitivity. The structural results of the current x-ray study correct predictions made in an early model building study regarding the binding of D-phenylalanine (Lipscomb, W. N., Hartsuck, J. A., Reeke, G. N., Jr., Quiocho, F. A., Bethge, P. H., Ludwig, M. L., Steitz, T. A., Muirhead, H., and Coppola, J. C. (1968) Brookhaven Symp. Biol. 21, 24-90).