Binding of ligands to the active site of carboxypeptidase A.

Rees, Douglas C.; Lipscomb, William N.

doi:10.1073/pnas.78.9.5455

Cited by 58 publications

(27 citation statements)

References 18 publications

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“…Crystallographic measurements performed on carboxypeptidase A showed that, unlike thermolysin, the guanidinium group of the arginine residue present in the active site strongly interacts with the free carboxyl group of substrates or inhibitors (35). Therefore, in a carboxydipeptidase such as ACE, it was assumed that the carbonyl group of the ultimate amide bond of substrates or inhibitors was hydrogen-bonded to an acceptor group of the enzyme backbone (33,36).…”

Section: Resultsmentioning

confidence: 99%

Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan.

Roques¹,

Lucas-Soroca²,

Chaillet³

et al. 1983

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

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Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyliglycine is a highly potent inhibitor (K; = 3.5 nM) of "enkephalinase," a metalloendopeptidase cleaving the Gly-Phe bond (positions 3 and 4) of enkephalins in brain tissue. In accordance with this property, thiorphan displays antinociceptive activity after systemic administration. However, thiorphan also inhibits to a lesser extent (K; = 140 nM) the widely distributed angiotensin-converting enzyme, a carboxydipeptidase implicated in blood pressure regulation. Therefore, in view of an eventual clinical use of enkephalinase inhibitors, it was very important to develop fully specific compounds. Such derivatives were obtained taking into account that N-methylation of the ultimate amide bond of dipeptides strongly decreases enkephalinase affinity without affecting angiotension-converting enzyme recognition, whereas retro-inversion of the amide bond leads to the inverse effect. Thus, the retro-inverso dipeptide (R)-H2N-CH(CH24)-NHCO-CH2-CO2H exhibits an inhibitory potency on enkephalinase (IC50 12 jM) close to that of the natural dipeptide L-Phe-Gly (IC50 3 jIM). This result shows the topological analogy between the crucial components involved in enkephalinase recognition both in active dipeptides and structurally related retro-inverso isomers. Taking into account these observations, retro-thiorphan, (R,S)-HS-CH2-CH-(CH20)-NHCO-CH2-COOH, was prepared. As compared to thiorphan, the retro isomer is 50% as potent (Ki = 6 nM) on enkephalinase but displays a drastic loss of potency on angiotension-converting enzyme (IC50 > 10,000 nM). This specificity was interpreted as a consequence of differences in the stereochemical constraints involving enzyme-inhibitor hydrogen bonding. This hypothesis is supported by reported crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. As expected, retro-thiorphan exhibits about the same analgesic potency as thiorphan on the hot plate and writhing tests in mice. Therefore, the topological concept of retro-inverso isomers could be extended to other enkephalinase inhibitors, allowing the design of potent and highly selective compounds occurring as new classes of analgesic and psychoactive agents.An important approach in the search for antinociceptive agents is to prevent degradation of the endogenous morphine-like peptides, enkephalins (1, 2), at the synaptic level. The characterization of a membrane.bound metalloendopeptidase called enkephalinase (3)(4)(5), which seems to be specifically involved in cleavage of the Gly-Phe (positions 3 and 4) bond of [Met]enkephalin (Tyr-Gly-Gly-Phe-Met) or [Leu]enkephalin (Tyr-GlyGly-Phe-Leu), made this approach possible. A highly potent enkephalinase inhibitor, thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]glycine [Ki = 3.5 nM (6, 7)], was rationally designed taking into account the occurrence in this peptidase of (i) a Zn atom in its catalytic site able to coordinate a thiol group with a strong affinity (7, 8); (ii) a Sj' subsite exhibiting a specificity for aro...

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Section: Resultsmentioning

confidence: 99%

Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan.

Roques¹,

Lucas-Soroca²,

Chaillet³

et al. 1983

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

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“…A. X-Ray studies have revealed that a glutamate carboxylate is also present in the active site of this enzyme (81), and the role of this carboxylate in catalysis is uncertain (82). The possible mechanisms wluch have been considered for carboxypeptidase are that the glutamate carboxylate acts as a general basic catalyst to activate a water molecule as a nucleophile or to serve as a nucleophile itself, thereby leading to the formation of a mixed anhydride reaction intermediate (83).…”

mentioning

confidence: 98%

Oxygen Chiral Phosphate Esters

Gerlt

Coderre

Mehdi

1983

Advances in Enzymology - And Related Areas of Molecular Biology

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“…Specific roles have previously been assigned to two of these residues: Arg-145 forms a salt bridge to the carboxylate group of ligands bound in the S' binding site (5), while Arg-71 hydrogen bonds to the carbonyl oxygen of the amino acid residue in the S2 binding subsite of the complex between CPase A and an inhibitory protein from potatoes (16). Significantly, Arg-127 is the only arginine of the three that does not form specific contacts to the inhibitor in this complex.…”

mentioning

confidence: 99%

“…Furthermore, from crystallographic studies of CPase A-ligand complexes, several pre-and posthydrolytic binding interactions between CPase A and substrates have been identified. The penultimate binding stage of peptide substrates is described, for example, as having the COO--terminal carboxylate group of the substrate bound to zinc (16 …”

mentioning

confidence: 99%

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Crystallographic studies on apocarboxypeptidase A and the complex with glycyl-L-tyrosine.

Rees¹,

Lipscomb²

1983

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

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The crystal structures of zinc-free carboxypeptidase A (apocarboxypeptidase A) and the complex of glycyl-L-tyrosine with apocarboxypeptidase A are described and compared to the corresponding structures of the zinc-containing enzyme. Only small conformational changes in the zinc ligands accompany removal of the metal. Interactions between the tyrosine residue of glycyl-L-tyrosine and apoarboxypeptidase A are similar to those observed in the complex with the holoenzyme. However, in the absence of zinc, the carbonyl oxygen of the glycyl moiety now receives a hydrogen bond from the side chain of arginine-127. Although not as yet observed, a similar shift of the carbonyl oxygen of a susceptible bond from the zinc to arginine-127 could stabilize tetrahedral intermediates generated during the hydrolysis of substrates by carboxypeptidase. (not the zinc) functions as the binding site for the carboxylate group of the peptide during the hydrolytic step. Instead, the zinc serves to polarize the carbonyl group of the susceptible bond and possibly as the source of a zinc-hydroxyl (or zinc water) species for nucleophilic attack on the substrate during a deacylation step (8,9).Removal of the zinc yields an inactive enzyme, apocarboxypeptidase A (apo-CPase A) (10). The conformation of apo-CPase A appears to be similar to that of the native enzyme (11). Zinc is bound quite rapidly by the apoenzyme, with concomitant recovery of activity. Moreover, the recombination of zinc with apo-CPase A is inhibited by peptides, suggesting that these ligands may bind to the apoenzyme (12). The location of bound peptides at the active site in apo-CPase A was demonstrated by difference Fourier studies at low resolution of the complex of apo-CPase A with the dipeptide glycyl-L-tyrosine (Gly-Tyr) (13).X-ray crystallographic methods are well suited for investigating the structural consequences of zinc removal on the active site conformation and ligand-binding properties of CPase A. Crystal structures of CPase A in the native state and complexed to a variety of ligands have been described (14-16). Direct removal of zinc from crystals of CPase A yields crystals of apo-CPase A that are isomorphous with those of the native enzyme (11). Consequently, sensitive difference Fourier techniques may be used to study the structural changes accompanying zinc removal from CPase A. In this paper, we compare the structures of apo-CPase A and the complex of Gly-Tyr with apo-CPase A to the corresponding forms of the zinc forms. The implications of these structures for the binding and cleavage of substrates by CPase A are discussed. MATERIALS AND METHODSBovine CPase A, (Cox), 8-hydroxyquinoline-5-sulfonate (HQSA), and Gly-Tyr were purchased from Sigma and used without further purification. Crystals of CPase A were grown by dialysis of CPase A solutions against 0.2 M LiCl/0.02 M Tris-HCl, pH 7.5, at 40C (11). apo-CPase A was prepared by the standard procedure of soaking the crystalline enzyme in a solution containing chelator (17, 18). Although o-phenanthrol...

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Binding of ligands to the active site of carboxypeptidase A.

Cited by 58 publications

References 18 publications

Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan.

Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan.

Oxygen Chiral Phosphate Esters

Crystallographic studies on apocarboxypeptidase A and the complex with glycyl-L-tyrosine.

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