Arginine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme

Carboxypeptidase H (CPH) is one of the later enzymes in the cascade of proteolytic steps required for the posttranslational processing of peptide hormone precursors, including processing of proenkephalin. In this study, CPH activity in the soluble and membrane fractions of enkephalin-containing bovine chromaffin granules was competitively inhibited by its products arginine and lysine. Ki values for arginine and lysine were 4.6 +/- 1.3 and 7.6 +/- 1.9 mM, respectively, indicating that arginine was a more effective inhibitor than lysine. Other amino acids (at 10 mM) had no effect. The in vivo intragranular concentrations of lysine and arginine are similar to the measured Ki values, indicating that product inhibition of CPH by basic amino acids may occur in vivo.     

Principles of hydroxamate inhibition of metalloproteases: carboxypeptidase A

Hydroxamic acids of structure RCON(OH)CH(2)CH(CH(2)C(6)H(5))CO(2)H induce micromolar competitive inhibition of catalysis for the enzyme carboxypeptidase A. Enzyme affinity depends on the nature of the acyl group, for RCO equaling HCO, CH(3)CO, FCH(2)CO, F(2)CHCO, F(3)CCO, CH(3)OCH(2)CO, or CH(3)OCO. In acid dissociation these residues yield hydroxamic acid pK(a) values that vary from 7.6 to 10.3. Profiles of inhibitory pK(i) plotted versus pH indicate characteristically a maximum effectiveness near neutrality. Weaker binding to enzyme is generally displayed in either acidic or alkaline solution, with the position of the alkaline limb of the profiles depending on the pK(a) of the inhibitor. A reverse-protonation pattern of association with the enzyme is indicated, in which the hydroxamate anion of the inhibitor displaces a relatively acidic H(2)O ligand (pK(a) of 6) from the active-site zinc ion of carboxypeptidase A. The metal-coordinating, N-substituted hydroxamic acid functional groups exist in solution as a mixture of syn and anti rotamers, with relative abundances that depend on their pK(a). A pyrrolidinone analogue having a conformationally syn-fixed cyclohydroxamic acid was not an especially potent inhibitor. Structure-activity relationships suggest design criteria for hydroxamic acid inhibitors in order to provide most effective binding with metalloenzymes.     

Stereoselective inhibition of glutamate carboxypeptidase by chiral phosphonothioic acids

A series of phosphonothioic acid derivatives of (S)-2-hydroxyglutarate with various alkyl or aryl ligands to the central phosphorus atom was examined for stereoselective inhibition of the glutamate carboxypeptidase, carboxypeptidase G. The inhibitory potencies of these stereoisomers were compared to corresponding synthetic phosphonic acid analogues in order to reveal the significance of the sulfur ligand of the phosphonothioic acid motif upon the inhibition of this metallopeptidase. The acquisition of the individual phosphonothioic acid diastereomers was achieved through the resolution of the respective phosphonate ester precursors. In all cases, the (+)p-diastereomers of these phosphonothioic acid derivatives of (S)-2-hydroxyglutarate were found to be more potent inhibitors of glutamate carboxypeptidase than the corresponding (-)p antipodes with the most dramatic difference observed for the butyl isomers (13.6-fold). Based upon Ki values obtained, the most potent inhibitor of the series by nearly an order of magnitude was the (+)p-n-butylphosphonothioic acid derivative, revealing specific structural and stereochemical requirements by this glutamate carboxypeptidase. With the exception of the (+)p-n-butyl analogue, the isosteric replacement of oxygen with sulfur of the phosphonic acid moiety did not enhance inhibitory potency.     

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.     

Mechanistically significant diastereoselection in the sulfoximine inhibition of carboxypeptidase A

The diastereomers of L-alpha-[ [S-(2-phenylethyl)sulfonimidoyl]methyl]benzenepropanoic acid bind differentially to carboxypeptidase A. These putative transition state-analogue inhibitors show unique and interpretationally significant pH dependences for Ki, as well as for the visible absorption spectra of their E.I complexes in the case of the cobalt-substituted enzyme. From the geometry of the enzymically preferred isomer, it may be concluded that the mechanism of peptide scission by the enzyme entails addition of a nucleophile to the si face of the bound-substrate prochiral carboxamide linkage. New interpretational constraints on the mode of action of the enzyme are thereby imposed.     

Synergistic inhibition of carboxypeptidase A by zinc ion and imidazole

Zinc ion in solution yields a 560-fold enhancement in the kinetic inhibition of carboxypeptidase A by the simple heterocycle imidazole, behavior attributed to formation of a ternary complex of the three species. However, the effect is partially negated by formation of less-inhibitory Zn2+(C3H4N2)2-4 coordination complexes, providing for the enzyme an anomalous profile of catalytic rate versus imidazole concentration.     

Product stabilization of the two forms of carboxypeptidase A from buffalo pancreas

The two forms of buffalo carboxypeptidase A lose a part of their activity when incubated at their optimal temperature. Both the forms are protected from heat denaturation by L-phenylalanine, one of the reaction products while the other reaction product hippuric acid provides only limited protection. It has also been shown that L-phenylalanine and hippuric acid bind at the same site of the enzyme and that the release of L-phenylalanine follows that of hippuric acid. On this basis, a new mechanism for the hydrolysis of acyldipeptides by carboxypeptidase A has been proposed     

Stereoselective inhibition of glutamate carboxypeptidase by organophosphorus derivatives of glutamic acid

A series of alkyl and aryl phosphonyl, thiophosphonyl, and dithiophosphonyl derivatives of (S)- and (R)-glutamic acid were prepared and examined for inhibitory potency against glutamate carboxypeptidase (carboxypeptidase G). The acquisition of the phosphonamidodithioic acids and the individual phosphonamidothioic acid diastereomers was achieved through a common phosphonamidothiolate precursor, which also allowed for the chromatographic resolution of the chiral phosphorus center of the phosphonamidothioic acids. The most potent inhibitor of the series was the n-butylphosphonamidate derivative of the natural isomer of glutamic acid. Although each diastereomeric pair of three phosphonamidothionates exhibited stereoselective inhibition consistent with the configuration of the chiral phosphorus center, this effect was generally not remarkable. More important, was the effect of carbon stereochemistry upon glutamate carboxypeptidase inhibition as exemplified by a limited series of enantiomeric pairs of phosphonamidate and phosphonamidodithionate derivatives of glutamic acid. The phosphonamidate analogs derived from the unnatural stereoisomer of glutamic acid were devoid of inhibitory potency in contrast to their enantiomers. Surprisingly, the phosphonamidodithionates derived from the unnatural stereoisomer of glutamic acid demonstrated greater inhibitory potency than their naturally-derived antipodes.     

Kinetics and inhibition of glutamate carboxypeptidase II using a microplate assay

Proteomics, the study of protein function on a global scale, will play an important role in furthering our understanding of gene functions, complex biological pathways, and discovery of novel drug targets. A number of techniques have been developed for proteomic studies to identify and analyze proteins, compare protein expression levels, and study protein-protein interactions. Recent developments have applied a DNA array-type approach to immobilize proteins on a surface for high-throughput analysis. Here we report the development and construction of protein chips using derivatized glass and nitrocellulose-coated slides and the employment of recombinant proteins fused with green and red fluorescent proteins for detection. Fluorescent signals were found to be proportional to the amount of arrayed proteins and could be readily detected with a conventional fluorescence slide scanner. This technique allows the investigation of protein-protein interactions without the need for additional labeling steps of probe proteins.     

Substrate inhibition in the hydrolysis of N-acylglycine esters by carboxypeptidase A

The rates of hydrolysis of a series of 21 N-acylglycine esters (YCONHCH2CO2CH(CH2CH3)CO2H (2)) by bovine pancreatic carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) have been studied over the substrate concentration range 10(-4)-10(-1) M at pH 7.5, 25 degrees C, ionic strength 0.5. All substrates display substrate inhibition except Y = CH3, CH3CH2 and (CH3)3C for which normal Michaelis-Menten kinetics are observed. In all cases substrate inhibition is consistent with the formation of an ES2 complex and parameters for the second-degree rate equation v/E = (kapp2 S + kapp3 S2/KappSS)/(KappS + S + S2/KappSS) have been evaluated. For a series of eight aliphatic groups varying in size between Y = CH3 and Y = cyclo-C6H11 the following linear correlations were observed: -log KappS = 0.82 pi + 1.32 and log kapp2/KappS = 0.71 pi + 5.81 (pi is Hansch's hydrophobicity parameter). Aryl and aralkyl Y moieties deviate from these correlation lines. KappSS also depends on the hydrophobicity of Y but no quantitative correlation is obvious. Thus the Y unit of 2 is involved in a hydrophobic interaction with the enzyme when 2 binds at both the catalytically productive and inhibitor sites. Parameters for the enzymic hydrolysis of the esters YCONHCH2CO2CH(CH2CH(CH3)2)CO2H (3) (Y = C6H5(CH2)n (n = 0, 1, 2)) are also presented. Pronounced nonproductive 1: 1 enzyme.substrate complex formation is observed for each of 2: Y = C6H5(CH2)n (n = 2, 3) and 3: Y = C6H5(CH2)2. Hippurate anion is shown to be an uncompetitive inhibitor (Ki = 12 mM) for the hydrolysis of 2: Y = (CH3)3C. Data are now available which can only be interpreted in terms of at least three enzymic sites being available for hydrophobic interactions with ester substrate molecules.     

Product inhibition in sludge digestion

Hydrogen and acetate inhibit the microbial degradation of propionate and ethanol in digesting sludge. Propionate oxidation is more sensitive than ethanol oxidation to product activities. This difference can be explained on thermodynamic grounds. It is concluded that the limiting factors for complete anaerobic mineralization of biodegradable organic matter in digesting sludge are to be sought in the boundary conditions for the exergonic oxidation of propionate. The different ecological significances of hydrogen and acetate as inhibitory agents of fatty acid degradation in sludge digestion are discussed. Hydrogen partial pressure and alkalinity are recommended as parameters in monitoring sludge digestion.     

Nitro as a novel zinc-binding group in the inhibition of carboxypeptidase A

2-Substituted 3-nitropropanoic acids were designed and synthesized as inhibitors against carboxypeptidase A (CPA). (R)-2-Benzyl- 3-nitropropanoic acid showed a potent inhibition against CPA (K(i)=0.15 microM). X-ray crystallography discloses that the nitro group well mimics the transition state occurred in the hydrolysis catalyzed by CPA, that is, an O,O'-bidentate coordination to the zinc ion and the two respective hydrogen bonds with Glu-270 and Arg-127. Because the nitro group is a planar species, we proposed (R)-2-benzyl-3-nitropropanoic acid as a pseudo-transition-state analog inhibitor against CPA.     

Bioanalysis of N-acetyl-aspartyl-glutamate as a marker of glutamate carboxypeptidase II inhibition

We report the characterization of two methods for the analysis of N-acetyl-aspartyl-glutamate (NAAG) in biological fluids. In the first method, NAAG concentrations were calculated based on differences between glutamate concentrations before and after NAAG hydrolysis with exogenous glutamate carboxypeptidase II (GCP II) using high-performance liquid chromatography (HPLC) followed by fluorescence detection. In the second method, NAAG levels were quantified directly using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analyses of NAAG levels in human cerebrospinal fluid samples using either method gave similar results within experimental error, confirming the validity of the two independent measurements. These methods will be useful in future clinical trials to assess drug-induced GCP II inhibition in biological matrices.     

Competitive inhibition of a glutamate carboxypeptidase by phosphonamidothionate derivatives of glutamic acid.

Several N-thiophosphonyl-glutamates were found to be potent competitive inhibitors of a zinc-dependent glutamyl hydrolase, carboxypeptidase G (CPG). Weak inhibition exhibited by an analogous N-phosphonyl-glutamate suggests that the enhanced potency of the phosphonamidothioates is due to the presence of their sulfur ligand and its favorable interactions with active site features, presumably zinc(II).     

Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.     

Highly sensitive high-performance liquid chromatography-fluorimetric assay method for carboxypeptidase H activity

A rapid and sensitive high-performance liquid chromatographic (HPLC)-fluorimetric assay method has been developed for the determination of carboxypeptidase H activity based on the measurement of N-(5-dimethyl-aminonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically from dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyl-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slower with an acidic buffer at pH 4.6. The new HPLC method separates the product and substrate in less than 5 min using an elution buffer at pH 7.0 containing 60% methanol. Using this method carboxypeptidase H activity has been detected in rat sciatic nerves. This HPLC method facilitates the assay of carboxypeptidase H activity in the enzyme samples from various tissues.