Differences between the Conformation of Arsanilazotyrosine 248 of Carboxypeptidase A in the Crystalline State and in Solution

Abstract

Coupling of carboxypeptidase A crystals with diazotized arsanilic acid specifically labels tyrosine 248, an active-site residue of the enzyme. Many azophenols are yellow and their zinc complexes are red; the "yellow" absorption spectrum of zinc arsanilazocarboxypeptidase crystals is characteristic of the arsanilazotyrosyl group, not of the zinc complex. This is consistent with the interpretation of x-ray data on native crystals of carboxypep, tidase A, indicating that tyrosine 248 and the zinc atom are too far apart to form a complex. However, the enzyme in solution is red, denoting the formation of a complex between zinc and arsanilazotyrosine 248. The most likely interpretation of the data is that the orientation of arsanilazotyrosine 248 in solution and in the crystal is different. If the unlabeled tyrosine 248 of native carboxypeptidase undergoes similar changes, these data may bear upon the low activity of the enzyme in the crystalline state and on the catalytic mechanism of the enzyme based on the crystal structure. The opportunities for analogous spectrochemical studies of other, similar systems are pointed out.The increasing importance of x-ray crystallography in studies of structure-function relationships of enzymes has generated considerable interest in the effect of crystallization on conformation. Knowledge of the relative conformations of enzymes in the solid state and in solution is now beginning to accumulate (1). For example, the properties of carboxypeptidase have been studied in particular detail from this point of view (2-4). The specific activity of carboxypeptidase in the crystalline state is only about 0.3% of that in solution. Further, the two physical states differ in the ease of zinc removal and in the enzymatic consequences of inorganic and organic modifications, though the basis of these phenomena has remained obscure. Hence, search for additional effects of the two states on conformation seemed indicated.We have emphasized the value of spectrochemical probes in the assessment of changes in local protein conformation. The coupling of tyrosyl residues with diazonium salts has proven particularly useful in this regard (5, 6). We have now used diazotized arsanilic acid for the specific modification of tyrosine 248, an active-site residue of carboxypeptidase A (7 METHODSA 50-fold molar excess of freshly prepared diazotized parsanilic acid was added to a suspension of carboxypeptidase crystals in 0.01 M KHCO3, pH 8.8, 00C and, after 3 hr, the reaction was stopped by centrifugation. The modified crystals were washed five times with metal-free water, suspended in 0.05 M Tris -HCl (pH 8.2), and fractionated as described by Richards et al. (2). Both the quantitation of amii)o-acid residues modified and the preparation of apoenzyme have been described (9). Tetrazolylazo-N-carbobenzoxytyrosine was prepared as described (10). Circular dichroic spectra were obtained with a Cary model 60 spectropolarimeter and absorption spectra with a Cary model 14R recording spectrophotometer. The absorpt...

Effects of pH on the structure and function of carboxypeptidase A: crystallographic studies.

“…The main point of this study is that at any of these pH values (7.5-9.5) we find no evidence for a conformation of Tyr-248 in the down position, which is observed upon binding of analogs and inhibitors (2,19,20,31), or for a conformation in which the phenolic group is directly bound to the Zn atom ("Zn-bound Tyr-248" conformation), as suggested from spectroscopic data of CPA derivatives in which the Tyr-248 side chain had been chemically modified (7)(8)(9). A more direct approach, in which we started with Tyr-248 in both the up and Zn-bound positions, resulted in least-squares refinement of occupancies of <5% in the Znbound position and -100% in the up position.…”

“…The data presented above strongly indicate that Tyr-248 is relatively constraint free in the crys- tal form used in our studies (22). We therefore conclude that since no Zn-bound Tyr-248 is observed at any pH between 7.5 and 9.5, this conformation does not exist, or it is insignificantly populated at this pH range, implying that coordination of the phenolic hydroxyl is not an important feature of the resting enzyme and is definitely not a required conformation for activity of CPA, as has often been suggested (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).…”

“…As for the location of the chemically modified side chain of Tyr-248 in arsanilazo-CPA and [248-o-nitrotyrosine]CPA (nitro-CPA), it is quite possible that the predominant conformation of this residue is that referred to above as the Znbound Tyr-248 conformation (8,9,18) or other altered conformations in which the phenolic group is brought to close proximity with a positive charge (13,14), both in the crystal and in solution. However, considering the crystallographic results presented above, we conclude that direct bonding of Tyr-248 to Zn (or any other altered conformation) is favored by the chemical modification itself and that this conformation is neither a common nor a required feature for the active enzyme.…”

“…In a study of the three-dimensional structure of the complex of CPA with the 39-amino acid inhibitor from the potato (PCI), it was shown that cleavage has taken place and that Tyr-248 donates a hydrogen bond to the newly foftned carboxylate anion and receives a hydrogen bond from the originally penultimate peptide bond (20). Whether Tyr-248 has other roles such as a proton donor in the cleavage reaction (2)(3)(4)(5) or whether formation of a bond between Tyr-248 and Zn is an essential step (8,9) in the activity of CPA are open to question.…”

“…rectly bound to the Zn atom ("Zn-bound Tyr-248" cohformation) in the native unliganded enzyme, and that movement of Tyr-248 away from the Zn is an essential step in catalysis of the unmodified enzyme as substrates bind (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)). In contrast, the crystallographic studies indicate that in the unmodified enzyme the side chain oxygen of Tyr-248 is about 17 A away from the Zn (1, 2) (the "up" position) and that the phenolic oxygen moves about 12 A toward the active site as substrates bind (2,19) (the "down" position).…”