Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

(R)-2-Benzyl-5-cyano-4-oxopentanoic acid (compound 4) was studied as a mechanism-based inactivator (suicide substrate) for the zinc protease carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1). This compound was designed rationally based on the knowledge of the active site topology and the reported stereospecific proton exchange on ketonic substrate analogue (R)-3-(p-methoxybenzoyl)-2-benzylpropanoic acid [Sugimoto, T. & Kaiser, E. T. (1978) J. Am. Chem. Soc. 100, 7750-7751] by CPA. It is suggested that enzymic deprotonation on the C-5 methylene moiety may result in the transient formation of a ketenimine as the key intermediate that partitions between turnover and enzyme inactivation. The enzyme inactivation exhibited pseudo-first-order kinetics, was irreversible, and could be fully prevented in the presence of the reversible inhibitor benzyl-succinate. The inactivation rate constant, kintact, was evaluated to be 0.083 +/- 0.003 min-1 and kcat was measured at 1.78 +/- 0.06 min-1. In turn, a partition ratio of 28 +/- 3 was calculated. The reversible inhibitor constant (Ki) was measured at 1.8 +/- 0.5 microM, indicative of a high affinity for compound 4 shown by CPA; however, Km for the turnover process was determined at 4.93 +/- 0.43 mM. Kinetic analysis and labeling by the radioactive form of the inactivator suggested that the stoichiometry for protein modification by compound 4 approaches a 1:1 ratio.
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PMID:Design of an effective mechanism-based inactivator for a zinc protease. 230 May 47

The structure of the complex between carboxypeptidase A alpha (EC 3.4.17.1) and the ketonic substrate analogue 5-benzamido-2-benzyl-4-oxopentanoic acid (BOP) has been determined by x-ray crystallographic methods to a resolution of 1.7 A (final R = 0.191). Interestingly, BOP was observed to bind to the active site of carboxypeptidase A alpha as the covalent hydrate adduct. Because BOP is probably less than 0.2% hydrated in aqueous solution, this result was unexpected. One possibility is that the zinc-bound water of the native enzyme added to the ketone carbonyl. Alternatively, the enzyme may preferentially scavenge the hydrated ketone as it is continuously maintained at equilibrium in the solution in which the carboxypeptidase A alpha crystals were immersed. In either case, this mode of binding of BOP to carboxypeptidase A alpha provides an example of the preferred binding of a model of a structure along the reaction coordinate of a hydrolytic reaction.
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PMID:Mechanism of carboxypeptidase A: hydration of a ketonic substrate analogue. 347 Jul 37

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