Conformations of Arsanilazotyrosine - 248 Carboxypeptidase A 0 , , s , . Comparison of Crystals and Solution ( intramolecular coordination / circular dichroism and absorbance - pH titration )

The spectra of the a, ,, and -y forms of zinc monoarsanilazotyrosine-248 carboxypeptidase A are indistinguishable. At pH 8.2 their crystals are yellow, while their solutions are red, Xmax 510 nm. Absorption and circular dichroism-pH titrations of the modified zinc and apoenzymes demonstrate that the absorption band at 510 nm is due to a complex between arsanilazotyrosine248 and the active-site zinc atom. Two pKapp values, 7.7 and 9.5, characterize the formation and dissociation of this arsanilazotyrosine-248-Zn complex. On titrations of the apoenzyme, the absorption band at 510 nm is completely absent at all pH values. Instead, there is a single pKapp, 9.4, due to the ionization of the azophenol, Xxs 485 nm. Substitution of other metals for zinc results in analogous intramolecular coordination complexes with absorption maxima and circular dichroism extrema characteristic of the particular metal. Similar data and conclusions have been derived from studies of heterocyclic azophenol metal complexes. The present studies demonstrate that the conformation of the crystals of all generally available a, A, and My forms of the arsanilazoenzyme differs from that of their solutions. The spectra of the modified x-ray crystals, however, differ from those of all other carboxypeptidase forms and crystal habits studied. The internal consistency of their data, their interpretation, and the conclusions ofLipscomb and coworkers [Proc. Nat. Acad. Sci. USA (1972) 69, 28502854] are examined. Dissimilar chemical modification or conformation is thought to underlie these differences. The arsanilazotyrosine-248-zinc complex is a sensitive, dynamic probe of environmental conditions. Its response to changes in pH and physical state of the enzyme suggest different orientation of the arsanilazotyrosine-248 side chain in solution from that in the crystal. This finding calls for reexamination of the basis of the substrate-induced conformation change which has been thought to be critical to the mechanism, postulated on the basis of the x-ray structure analysis performed at pH 7.5. The integration of functional data obtained in solution with the structure derived from crystals remains one of the important problems in discerning the mechanism of action of an enzyme. We have searched for means that in solution could simultaneously gauge activity and the structural dynamics of the active center under a wide range of environmental conditions. Several chromophoric probes have been found particularly helpful in this regard (1, 2). In relating the specific structure of carboxypeptidase Aa EC 3.4.2.1 to the catalytic mechanism of carboxypeptidase in general, the location of Tyr-248 with respect to the active-site Zn atom has been thought critical (3, 4). When specifically coupled with diazotized arsanilic acid, solutions of carboxypeptidase Ay exhibit an absorption spectrum with a maximum at 510 nm (red) thought to be indicative of an intramolecular coordination complex between mono-arsanilazotyrosine-248 and Znt. This complex can be dissociated and its accompanying spectrum can be abolished by crystallization of the enzyme, removal of the metal, or interposition of substrates or by inhibitors (2). We have now examined the absorption and circular dichroism spectra of all three forms, a, fl, and y, of zinc arsanilazotyrosine-248 carboxypeptidase. Their crystals are all yellow, and their solutions all exhibit the same characteristic red absorption maximum at 510 nm, as observed previously for the -y enzyme (2, 5). Moreover, the pH dependence of this spectrum provides detailed information regarding the molecular basis of its origin. These data demonstrate that the orientation and mutual proximity of azotyr-248 and Zn in the crystals of a, A, and y azocarboxypeptidase differ from that of their solutions, as reported earlier (1, 2, 5). Recently, Lipscomb and coworkers (4), using conditions and forms of the enzyme comparable to ours, i.e., crystals of Aa and Ay elongated along the b-axis, successfully repeated these experiments and obtained identical spectral results; the spectra of the crystals differed from those of the solutions. However, using the unusual crystalline form of carboxypeptidase A., elongated along the a-axis which served for x-ray analysis, the same spectra were found for the crystals and solution. This has raised some important questions: Is the molecular basis of the 510-nm spectrum the same or different for the various modified carboxypeptidase forms? Alternatively, are the crystal structures of these various enzyme forms having different crystal habits the same or different? The present work raises doubts about the interpretation and conclusions of Lipscomb and coworkers (4). t To simplify nomenclature, zinc carboxypeptidase A (the zinc azoenzyme) and apoazocarboxypeptidase A (the apoazoenzyme) are used interchangeably with zinc monoarsanilazotyrosine-248 carboxypeptidase and apoarsanilazotyrosine-248 carboxypeptidase, respectively, of any enzyme form. Carboxypeptidase Ak used for x-ray structure analysis and with a crystal habit elongated along the a axis (4) was designated the x-ray crystal or enzyme. Azo-Tyr-248 refers to mono-arsanilazotyrosine-248, the azophenolate ion to its ionized species. The absorption spectrum of zinc monoarsanilazotyrosine-248 carboxypeptidase is defined as "yellow" in the absence of an absorption band at X,. 510 nm, as "red" in its presence. 2006 * To whom correspondence should be addressed. PArsanilazotyrosine-248 Carboxypeptidase Aa,,s, 2007 MATERIALS AND METHODS Procedures to isolate, characterize, and crystallize the various forms of carboxypeptidase A and to prepare their arsanilazo derivatives have been published (2, 6-9). Apoenzymes were prepared by soaking crystals in 1,10-phenanthroline (Auld, D. S., to be published). The a and j# forms of carboxypeptidase are much more soluble than the y enzyme (10). Hence, different conditions, critical for labeling them with a single arsanilazotyrosine-248 residue per molecule (9) had to be used for each form and crystal type of the enzyme. Precautions to prevent contamination by adventitious metal ions (11) were taken throughout. The thermostated titration cell of Auld and French (12) was adapted to the Cary model 14R recording spectrophotometer.for pH titrations from pH 6.0-11.0. Zincor other metal-carboxypeptidase (and the corresponding apoenzyme), about 60 uM in 0.02 M Tris HCl-0.5 M NaCl buffer (pH 6.0) at 230 i 0.1 was titrated with aliquots of 0.1 N NaOH to result in pH increments of 0.2-0.4. The absorption spectrum between 300 and 650 nm was recorded after each addition of base. Circular dichroic spectra were obtained with a Cary model 61 spectropolarimeter. Measurements between 300 and 650 nm were performed in 1-cm cells at protein concentrations of 30-60 ,uM. The pH dependence of the circular dichroic spectra of zinc and apoazocarboxypeptidase was also determined from pH 6.5-10.8 by stepwise addition of 0.1 N NaOH to 3-ml samples. The pH was measured with a CK 2321 Radiometer (Copenhagen) electrode before and after each spectrum was recorded. Mono-tetraazolyl-N-carbobenzoxytyrosine was prepared as described (13) and its concentration was determined by use of 6416 = 4.39 X 103 M-1 cm-'. Spectrophotometric pH titrations of mono-tetraazolyl-N-carbobenzoxytyrosine and its zinc complex were performed as for the enzyme; 0.1 mM each of mono-tetraazolyl-N-carbobenzoxytyrosine and ZnSO4 *7 H20 was used. Theoretical titration curves were fitted to the experimental data by use of a nonlinear least squares program written for the Hewlett Packard 9810A calculator and kindly provided by Dr. Thayer French. RESULTS AND DISCUSSION The a, #, and y zinc azoenzymes, modified according to criteria described (2, 9), have identical spectra and activities. The crystals are yellow but turn red when dissolved at pH 8.2 as expected when azoTyr-248 and zinc form a complex (2, 5). Hence, the particular enzyme form used will be indicated only for comparative purposes. The red solution Xmax 510 nm (e = 8,000)1, changes to yellow on (a) crystallization, (b) removal of zinc, (c) addition of substrate, (d) addition of inhibitors, (e) denaturation of the enzyme, or (f) lowering of the pH below 6 (2). Spectral titration (Fig. 1A) of zinc azocarboxypeptidase generates an absorption maximum at 510 nm between pH 6.3 and 8.5. When the pH is raised to 10.8, the maximum shifts progressively to 485 nm (e = 10,500), typical for the free azophenolate ion. Significantly, over the lower pH range there is but one isosbestic point, at 428 nm, while over the higher range there are two, at 412 and 520 nm. These data provide direct evidence for the progressive formation of at least three 0