The pH-dependence of the non-specific esterase activity of carboxypeptidase A

Bunting, John W.; Kabir, Shaikh H.

doi:10.1016/0005-2744(78)90259-0

Cited by 3 publications

(3 citation statements)

References 23 publications

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“…It appears to be the main -tocopherol oxidation product on the basis of refractive index detection. Other a-tocopherol (23) Corey, E. J.; Albright, J. O.; Barton, A. E.; Hashimoto, S. J. Am.…”

Section: Analyzed Resultsmentioning

confidence: 99%

Autoxidation of model membrane systems: cooxidation of polyunsaturated lecithins with steroids, fatty acids, and .alpha.-tocopherol

Weenen¹,

Porter²

1982

J. Am. Chem. Soc.

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Section: Analyzed Resultsmentioning

confidence: 99%

Autoxidation of model membrane systems: cooxidation of polyunsaturated lecithins with steroids, fatty acids, and .alpha.-tocopherol

Weenen¹,

Porter²

1982

J. Am. Chem. Soc.

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“…CPA is known to be reasonably active only in the pH range of approximately 6-9, with an optimum around pH 7.5 (34). Several kinetic studies (35)(36)(37)(38)(39)(40)(41)(42)(43) relate to the details of pH-activity relationships in the hydrolysis catalyzed by CPA and contain attempts to assign the usually observed high (z9) and low (-6-6.5) pKs to specific catalytic groups (44,45). Although these kind of pK assignments are usually complicated and may be misleading (46), we present here a limited discussion.…”

Section: Discussionmentioning

confidence: 99%

“…pH profiles (40)(41)(42)(43). The low pK could be assigned to the ionization of either Glu-270 (47,48) or the Zn-bound water molecule (6,49), and the high pK could be assigned to the ionization of Tyr-248, Tyr-198, or the Zn-bound water (3,6,48).…”

Section: Discussionmentioning

confidence: 99%

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

Shoham¹,

Rees²,

Lipscomb³

1984

Proc. Natl. Acad. Sci. U.S.A.

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High-resolution crystal structures are described for carboxypeptidase A (EC 3.4.17.1) in crystals grown at pH 8.5, 9.0, and 9.5 and compared with the structure at pH 7.5. The comparison shows that in the pH range of 7.5-9.5 the enzyme structure is practically unchanged, and, most importantly, that the flexible side chain of Tyt-248 remains exclusively in the "up" position, awdy from the Zn atom, throughout the pH range. There is no evidence fdr binding of Tyr-248 to Zn at any of these pH values. We conclude that the interaction of Tyr-248 with Zn is not an essential part of the mechanism of carboxypeptidase A and that its occurrence is an artifact of chemical modification of Tyr-248. It is also suggested that Tyr-248 is not uniquely associated with the observed high pK of the enzymatic hydrolysis.Carboxypeptidase A (CPA; peptidyl-L-amino acid hydrolase, EC 3.4.17.1) catalyzes the hydrolysis of the carboxylterminal residue from peptide or ester substrates by cleavage of the peptide or ester bond. The three-dimensional structure is known to a resolution of 1.54 A (1). Various mechanisms for the activity of CPA toward substrates have been proposed (2) and recently reviewed (3-7). Of the potential catalytic groups that are in the region of substrate in the x-ray diffraction studies [Glu-270, L3-Zn-OH2 (in which L is a protein ligand), H20, and Tyr-248], we examine here the possible roles of Tyr-248.Studies of spectral changes of [248-arsanilazotyrosine]-CPA (arsanilazo-CPA) as a function of pH (8, 9) have indicated that the phenolic oxygen of the modified Tyr-248 is bound to the Zn in the unliganded enzyme. On the bdsis of the results of these studies (8, 9) and related ones (10-18) it has been suggested that the phenolic group of Tyr-248 is directly 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 (8-18). 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). Moreover, a study at 1.5-A resolution shows that at pH 7.5 there is no observable binding of Tyr-248 to the Zn in the unmodified enzyme (1). 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 thatTyr-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 (2-5) 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. In this paper we report a crystallographic study that extends the earlier study of the nati...

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Carboxypeptidase A mechanisms.

Lipscomb¹

1980

Proc. Natl. Acad. Sci. U.S.A.

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The mode of binding of a ketonic substrate, which is an analogue of esters in which the 0 of the scissile bond is replaced by CH2, to carboxypeptidase A is similar to that of Gly-Tyr. The site is Si, with the side chain in the pocket of the enzyme, the carboxylate salt-linked to , and the carbonyl group bound to Zn. Thus, esters are probably cleaved at the peptide cleavage site, although not necessanly with the same rate-controlling step or by the same detailed mechanism. The large differences found between the behavior of the enzyme in solution and in one crystalline phase do not apply to a different crystalline phase. For those enzymes for which x-ray diffraction studies have been made on the three-dimensional structures of complexes with ligands, a new level of mechanistic questions arises, related to the detailed succession of binding and catalytic steps. The structures of the long-lived complexes of Gly-Tyr, Phe-GlyPhe-Gly, and other ligands with carboxypeptidase A, for example, along with a wealth of other chemical evidence, have led to detailed proposals (1-4) for the mechanisms of cleavage of peptides and esters. The principle of charge neutrality has been used to treat the general acid-base catalyzed or, alternatively, nucleophilic, first step, and to discuss possible deacylation steps (5).In this paper, the relationship between structural studies and more recent biochemical studies is discussed, with emphasis on the ambiguities in the mechanisms of action of this enzyme.Mechanistic deductions using x-ray results Hydrolytic cleavage of the COOH-terminal peptide bond of the substrate involves nucleophilic attack at the carbonyl carbon and electrophilic attack at the amide NH of this bond. Candidates for nucleophilic attack are Glu-270 to form an anhydride, Glu-270-promoted attack by H20, or (with considerable substrate distortion) a Zn-bound H20 or OH-which is not present in the initial complexes of the x-ray diffraction studies. Candidates for proton donation are Tyr-248 or, less likely, H20. The Zn binds to the carbonyl oxygen of these substrates and initially serves to polarize this C=O bond. If an anhydride is formed, deacylation could occur by attack of Zn-bound water or hydroxyl ion (3,5).The productive binding site S1 places the COOH-terminal side chain in a pocket of the enzyme and binds the substrate's COOH-terminal carboxylate group by two hydrogen bonds to Arg-145 (Fig. 1). Extension of this site shows Arg-127 (SI) 4 A away from Arg-145 and Arg-71 (S2) an additional 4 A away near . This last region may be a recognition site initiating a sliding mechanism for movement of substrate into the pocket (6, 7) and is probably the site of kinetic anomalies for N-acyl-dipeptides (1). The extended binding site is five amino acids in length (8, 9), and it is known that longer substrates do not show these kinetic anomalies (8, 9).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S...

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The pH-dependence of the non-specific esterase activity of carboxypeptidase A

Cited by 3 publications

References 23 publications

Autoxidation of model membrane systems: cooxidation of polyunsaturated lecithins with steroids, fatty acids, and .alpha.-tocopherol

Autoxidation of model membrane systems: cooxidation of polyunsaturated lecithins with steroids, fatty acids, and .alpha.-tocopherol

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

Carboxypeptidase A mechanisms.

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