Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
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Electronic absorption, circular dichroic (CD), and magnetic circular dichroic (MCD) spectra have been determined for complexes of cobalt(II)-substituted carboxypeptidase A and five reversible inhibitors. Three of the inhibitors, N-(1-carboxy-5-butyloxycarbonylaminopentyl)-L-phenylalanine, (I); (R,S)-2-benzyl-4-oxobutanoic acid, (III); and 2-benzyl-4-oxo-5,5,5-trifluoropentanoic acid, (IV) are mechanism-based inhibitors. Another, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine, (II), is a tight binding, slowly hydrolyzed substrate. The fifth, phosphoramidon, (V), is a mechanism-based inhibitor of thermolysin, and may also bind to carboxypeptidase in a mechanism-based mode. The absorption and CD spectra of the enzyme-inhibitor complexes all differ from the spectrum of the free enzyme and from each other. The MCD spectra indicate that the tetrahedral coordination geometry of cobalt, which is distorted in the free enzyme, is also distorted in the inhibitor complexes, although to various degrees. The complexes of I and III are spectrally similar despite being structurally dissimilar, and that of IV, whose structure resembles III, is spectrally distinct, indicating that I and III, but not IV, may perturb the metal in nearly the same way. The absorption spectrum of IV is identical to that, at high pH, of Co(II)carboxypeptidase in which Glu-270 has been modified by a carbodiimide reagent, possibly pointing to a common perturbation of this residue. The absorption and CD spectra of II are similar to those of the catalytic intermediate that precedes the rate-limiting step in peptide hydrolysis [D. S. Auld, A. Galdes, K. F. Geoghegan, B. Holmquist, R. Martinelli, and B. L. Vallee, Proc. Natl. Acad. Sci. USA 81, 4675-4681 (1984)]. Since II is a substrate, the steady-state bound species that it generates may therefore be a true productive intermediate rather than a nonproductive mimic of an intermediate. The spectra of the complexes with II and V differ considerably despite structural similarities. The negative CD ellipticity of the free enzyme is reversed in sign in the presence of V, a phenomenon previously observed with complexes of Co(II)carboxypeptidase and dipeptides. This resemblance may result from a similar interaction of cobalt with the phosphoramidate group of phosphoramidon and the N-terminal amine of dipeptides. The spectra of reversible, mechanism-based inhibitors permit general structural predictions about true intermediates but require caution when used for assigning precise conformation and ligands of bound catalytic species.
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PMID:Effects of mechanism-based reversible inhibitors on the metal environment of cobalt(II)carboxypeptidase A: an electronic spectral study. 274 19

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A. 409 28

The Ki's of three peptide ketone and three peptide alcohol inhibitors of carboxypeptidase A are compared with Ki's of their respective isosteric peptide substrates, N alpha-benzoyl-L-phenylalanine, N alpha-benzoylglycyl-L-phenylalanine, and N alpha-carbobenzoxyglycylglycyl-L-phenylalanine. For the isosteric ketone analogues of these substrates, the respective Ki's are as follows: (2RS)-2-benzyl-4-(3-methoxyphenyl)-4-oxobutanoic acid, 180 +/- 40 microM; (2RS)-5-benzamido-2-benzyl-4-oxopentanoic acid (V), 48 +/- 7 microM; (2RS)-2-benzyl-5-(carbobenzoxyglycinamido)-4-oxopentanoic acid (IX), 9 +/- 0.1 microM. For the alcohols derived by reduction of each of these ketones, Ki's are as follows: (2RS,4RS)-2-benzyl-4-(3-methoxyphenyl)-4-hydroxybutanoic acid, 190 +/- 10 microM; (2RS,4RS)-5-benzamido-2-benzyl-4-hydroxybutanoic acid (IV), 160 +/- 62 microM; (2RS,4RS)-2-benzyl-5-(carbobenzoxyglycinamido)-4-hy droxypentanoic acid (XI), 600 +/- 100 microM. Ki values for the competitive peptide ketone inhibitors decrease with increasing peptide chain length. This is consistent with the possibility of increased binding interaction between inhibitor and enzyme by simple occupation of additional binding subsites by adding more amino acid residues to the inhibitor. In contrast, the Ki values of the alcohols (competitive or mixed inhibition) increased or remain essentially unchanged with increasing chain length. Increasing the chain length of ketone inhibitor V to give IX decreases Ki by one-fifth. The Ki of ketone IX is also less than 1/30th the Ki of its isosteric peptide and almost 1/70th that of its isosteric alcohol, XI.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of carboxypeptidase A by ketones and alcohols that are isosteric with peptide substrates. 409 29