Effects of Acetonitrile-Water Mixtures on α-Chymotrypsin Catalyzed Dipeptide Synthesis

-Chymotrpysin (EC 3.4 21.1) was immobilized by deposition on celite and subsequent cross-linking with glutaraldehyde. The effects of different mixtures of aqueous buffer and acetonitrile on the immobilized preparation were evaluated using a dipeptide synthesis as model reaction. The initial reaction rate at 6-95% of water increased with increasing water content. The maximum yield of peptide had two maxima; the first one at 6% of water (92%) and the second one at 80% of water (39%). The presence of two maxima was due to severe enzyme inactivation at intermediate water contents (50-60%). The immobilisation procedure slowed the inactivation of -chymotrypsin. Cross-linked enzyme was inactivated to a lesser extent than both free enzyme and enzyme that had been deposited on celite. The increased resistance to inactivation was, however, not sufficient to make peptide synthesis attractive at intermediate water contents (50-60%). In order to obtain good peptide yields, low water contents (below 10%) should be used.     

Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).     

The influence of water on protease-catalyzed peptide synthesis in acetonitrile/water mixtures

Protease-catalyzed peptide synthesis in acetonitrile/water mixtures, containing 0-90% water, was investigated. alpha-Chymotrypsin, as well as thermolysin, were deposited on solid supports, prior to exposure to the reaction media. Peptide syntheses were performed using both a kinetically controlled process (chymotrypsin) and an equilibrium-controlled synthesis (thermolysin). The activity of chymotrypsin decreased at low water contents. However, at low water contents (1-10%) hydrolytic side reactions were suppressed and high yields of dipeptides were obtained. Optimal water content for the thermolysin-catalyzed reaction was 4-8%. The dipeptides produced were fully soluble in the reaction mixtures. High operational stability for alpha-chymotrypsin was obtained during 216 h of reaction.     

A lipoyl synthetic octadecapeptide of dihydrolipoamide acetyltransferase specifically recognized by anti-M2 autoantibodies in primary biliary cirrhosis.

Close to 95% of patients with established clinical, biochemical and histologic features of primary biliary cirrhosis (PBC) possess antimitochondrial M2 antibodies reacting with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We examined the ability of synthetic peptides of E2 to be recognized in ELISA by sera from patients with PBC and autoimmune-related disorders. Sera from 14 PBC M2+ patients, 1 PBC M2- patient, 5 non-PBC M2+ patients, and 6 patients with chronic active hepatitis were studied. Among the seven E2 synthetic peptides tested (namely peptides 87-119, 167-184, 169-202, 267-302, 456-477, 498-513 and 530-543), only peptide 167-184 used as OVA conjugate and prepared with lipoic acid (LA) located on lysine 173 (natural inner lipoyl-binding site) was recognized in direct ELISA by PBC M2+ sera. The conjugated peptide 167-184 LA was not recognized in direct ELISA by non-PBC M2+ sera or by sera from patients with chronic active hepatitis. The free peptide 167-184 LA inhibited the ELISA reaction of PBC antibodies to PDH and totally abolished the typical immunofluorescence reaction of PBC sera on rat kidney, stomach and liver, or human HEp-2 cell substrates. No inhibition of ELISA or immunofluorescence reaction was found with the other E2 fragments including peptide 167-184 without LA. Our results show that the lipoyl moiety forms an integral part of a dominant conformational epitope recognized by PBC sera. Inasmuch as the peptide 167-184 LA was not recognized by non-PBC sera in direct ELISA, it could be used as a valuable probe for PBC diagnosis.     

Synthesis of a membrane protein with two transmembrane regions.

A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent.     

Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media

A depeptide synthesis was drastically influenced by the reaction temperature, in the range from -30 degrees to 25 degrees C. This article shows the kinetic reasons of this effect. alpha-Chymotrypsin was immobilized on celite and used in four different water-miscible solvents containing small amounts of water-miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamide (Ala-NH(2)) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p-values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N-acetyl-L-phenylalanyl-L-ananinamide (AcPheAla-NH(2)) as substrates. The Michaelis-Menten parameters, K(m,app) and V(max), were determined for ester hydrolysis and dipeptide hydrolysis. Both K(m,app) and V(max) tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20%C compared with 84% at 25 degrees C. (c) 1995 John Wiley & Sons, Inc.     

Synthesis of peptides, catalyzed by ficin

Synthesis of a model peptide Z-Ala-Val-Gly-OH catalyzed by ficin in phosphate buffer was studied with water-soluble esters Z-Ala-OCH2CO-Gly-OH and Z-Ala-OCH2CO-NHC2H4SO3Na as carboxyl components. The maximal yield of the peptide was obtained at pH 8-9. The effect of temperature and concentration of reagents was studied in the model reaction.     

Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme

The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme.     

Protein-protein interaction in low water organic media

Trypsin either modified with polyethylene glycol or as a suspended powder was used to catalyze digestion of protein substrates in benzene in order to get insight into protein-protein interactions in water-immiscible organic media. Depending on whether suspended or soluble trypsin was used, catalysis was found to proceed differently. In the first case, the amount of water in the reaction mixture (up to 1% v/v) appeared to be critical, and adsorption of water from the reaction medium by the protein substrate allowed it to behave as a hydrophilic support material comparable to that involved in immobilized enzymes. In the latter case, the presence of an additional nucleophile was a prerequisite for catalysis to proceed, and thus both water and nucleophile concentrations had some influence on trypsin activity. Phe-NH(2) was the most potent nucleophile for proteolysis catalyzed by polyethylene glycol-modified trypsin in organic media containing 1-2% water (v/v). The organic solvent-soluble enzyme was found to bind reversibly to the protein substrate as a function of both extent of hydration of the reaction medium and time of incubation. The overall results strongly suggested that modified trypsin catalyzed peptide bond hydrolysis at the protein substrate-organic solvent interface. Peptide mapping of bovine insulin digest by reversed-phase high-performance liquid chromatography definitely showed that enzyme-catalyzed proteolysis did occur in organic solvents with a concomitant and significant transpeptidation reaction.     

Amidation of growth hormone releasing factor (1-29) by serine carboxypeptidase catalysed transpeptidation

The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C-terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1-29)-NH2, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction, where the C-terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C-terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C-terminal sequence of GRF(1-29)-NH2 is -Met-Ser-Arg-NH2 the model reactions Bz-Met-Ser-X-OH (X = Ala, Leu, Arg) + H-Arg-NH2----Bz-Met-Ser-Arg-NH2 + H-X-OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98% and 41%, respectively. With carboxypeptidase W-II and X = Arg a yield of no more than 72% could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz-Leu-Gln-Asp-Ile-Met-Ser-Ala-OH the amidated product could be obtained in a yield of 78%, using carboxypeptidase Y, the only other product being Bz-Leu-Gln-Asp-Ile-Met-Ser-OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1-28)-Ala-OH was synthesized by the continuous flow polyamide solid-phase method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urinary oxytocin as a non-invasive biomarker for neurohypophyseal hormone secretion

The objective was to characterize the urinary oxytocin (OT) system with the goal of using it as a biomarker for neurohypophyseal peptide secretion. We studied urinary OT secretion in mice under three conditions: (1) in OT gene deletion mice (OT -/-) which lack the ability to produce the peptide; (2) after arterial vascular infusion of OT and (3) after physiological stimulation with consumption of 2% sodium chloride. OT was measured by radioimmunoassay (RIA) and Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectroscopy (SELDI TOF MS). In OT -/- mice (n=25), urinary OT levels were not detectable, while in OT +/+ mice (n=23) levels were 250.2+/-35.3 pg/ml. To evaluate blood/urine transfer, mice with chronic carotid arterial catheters were infused with saline or OT (5 or 20 pmol/min). Peak urine OT levels were 89+/-11.5 and 844+/-181 ng/ml in the low and high OT groups, respectively. Proteomic evaluation showed MS peaks, corresponding to OT ( approximately 1009 Da) and a related peptide ( approximately 1030 Da) with highest levels in mice infused with OT. Salt loading (5 days of 2% NaCl as drinking water) increased plasma osmolality (3.3%), increased plasma and urinary vasopressin (AVP), but caused no changes in OT. Thus, using non-invasive urine samples, we document that urinary OT and AVP can be used to monitor changes in peptide secretion. Urinary OT and AVP, as well as other urinary peptides, may provide a viable biomarker for peptide secretion and may be useful in clinical studies.     

Silica, Alumina, and Clay-Catalyzed Alanine Peptide Bond Formation

The peptide bond formation of alanine (ala), ala + glycine (gly), ala + diglycine (gly₂), and ala + gly cyclic anhydride (cyc-gly₂) in drying/wetting cycles at 80°C was studied. Silica, alumina, and representative smectites—montmorillonite and hectorite—were used as catalysts, and the dependence of reaction yields on the available amount of water in the reaction systems was evaluated. Silica and alumina catalyze the formation of oligopeptide mainly in temperature fluctuation experiments, whereas higher amounts of water in the reaction system support clay-catalyzed reactions. Silica and alumina are much more efficient for amino acid dimerization than clays. Whereas only 0.1% of ala oligomerized on hectorite and no reaction proceeded on montmorillonite, about 0.9 and 3.8% alanine converted into its dimer and cyclic anhydride on silica and alumina, respectively. Clay minerals, on the other hand, seem to more efficiently catalyze peptide chain elongation than amino acid dimerization. The reaction yields of ala-gly-gly and gly-gly-ala from ala + gly₂ and ala + cyc-gly₂ reached about 0.3% on montmorillonite and 1.0% on hectorite. The possible mechanisms of these reactions and the relevance of the results for prebiotic chemistry are discussed. Received: 15 December 1996 / Accepted: 1 May 1997     

Self-association process of a peptide in solution: from beta-sheet filaments to large embedded nanotubes

Lanreotide is a synthetic octapeptide used in the therapy against acromegaly. When mixed with pure water at 10% (w/w), Lanreotide (acetate salt) forms liquid crystalline and monodisperse nanotubes with a radius of 120 A. The molecular and supramolecular organization of these structures has been determined in a previous work as relying on the lateral association of 26 beta-sheet filaments made of peptide noncovalent dimers, the basic building blocks. The work presented here has been devoted to the corresponding self-association mechanisms, through the characterization of the Lanreotide structures formed in water, as a function of peptide (acetate salt) concentration (from 2% to 70% (w/w)) and temperature (from 15 degrees C to 70 degrees C). The corresponding states of water were also identified and quantified from the thermal behavior of water in the Lanreotide mixtures. At room temperature and below 3% (w/w) Lanreotide acetate in water, soluble aggregates were detected. From 3% to 20% (w/w) long individual and monodisperse nanotubes crystallized in a hexagonal lattice were evidenced. Their molecular and supramolecular organizations are identical to the ones characterized for the 10% (w/w) sample. Heating induces the dissolution of the nanotubes into soluble aggregates of the same structural characteristics as the room temperature ones. The solubilization temperature increases from 20 degrees C to 70 degrees C with the peptide concentration and reaches a plateau between 15% and 25% (w/w) in peptide. These aggregates are proposed to be the beta-sheet filaments that self-associate to build the walls of the nanotubes. Above 20% (w/w) of Lanreotide acetate in water, polydisperse embedded nanotubes are formed and the hexagonal lattice is lost. These embedded nanotubes exhibit the same molecular and supramolecular organizations as the individual monodisperse nanotubes formed at lower peptide concentration. The embedded nanotubes do not melt in the range of temperature studied indicating a higher thermodynamic stability than individual nanotubes. In parallel, the thermal behaviors of water in mixtures containing 2-80% (w/w) in peptide have been studied by differential scanning calorimetry, and three different types of water were characterized: 1), bulk water melting at 0 degrees C, 2), nonfreezing water, and 3), interfacial water melting below 0 degrees C. The domains of existence and coexistence of these different water states are related to the different Lanreotide supramolecular structures. All these results were compiled into a binary Lanreotide-water phase diagram and allowed to propose a self-association mechanism of Lanreotide filaments into monodisperse individual nanotubes and embedded nanotubes.     

Enzymatic reactions in aqueous-organic media. VI. Peptide synthesis by α-chymotrypsin in hydrophilic organic solvents

The peptide synthesis from N-acetyl-L-tyrosine ethyl ester and amino acid amides was realized using α-chymotrypsin as a catalyst in ethanol or acetonitrile containing small amounts of water. In these reaction systems, the precipitates of phosphate salt, which was used as a component of buffer solution, are considered to act as carriers of chymotrypsin. It was found that peptide formation is competitive with hydrolysis of the substrate ester, but the secondary synthesis of the peptide from the hydrolysate was also considered to proceed. The yield of the peptide after 24 h reaction was strongly dependent on the water concentration; maximum yields of the peptide were obtained at water concentrations below 10% (v/v). The addition of tertiary amines, such as triethyl amine, markedly increased the peptide yield, probably due to the increase in the concentration of the nucleophilic amine components by neutralization of hydrohalides of amino acid amides. The effect of reaction temperature and the reactions with CT immobilized on PVA, chitosan, or TEAE-cellulose are also described.     

Distribution of G-proteins in rat liver plasma-membrane domains and endocytic pathways.

Cryoenzymology techniques were used to facilitate trapping an acyl-enzyme intermediate in beta-lactamase I catalysis. The enzyme (from Bacillus cereus) was investigated in aqueous methanol cryosolvents over the 25 to -75 degrees C range, and was stable and functional in 70% (v/v) methanol at and below 0 degree C. The value of kcat. decreased linearly with increasing methanol concentration, suggesting that water is a reactant in the rate-determining step. In view of this, the lack of incorporation of methanol into the product means that the water molecule involved in the deacylation is shielded from bulk solvent in the enzyme-substrate complex. From the lack of adverse effects of methanol on the catalytic and structural properties of the enzyme we conclude that 70% methanol is a satisfactory cryosolvent system for beta-lactamase I. The acyl-enzyme intermediate from the reaction with 6-beta-(furylacryloyl)amidopenicillanic acid was accumulated in steady-state experiments at -40 degrees C and the reaction was quenched by lowering the pH to 2. H.p.l.c. experiments showed covalent attachment of the penicillin to the enzyme. Digestion by pepsin and trypsin yielded a single labelled peptide fragment; analysis of this peptide was consistent with Ser-70 as the site of attachment.     

Aspartame precursor synthesis in water miscible cosolvent catalysed by thermolysin

Summary The synthesis of the dipeptideN-benzyloxycarbonyl-L- aspartyl-phenylalanine methyl ester, aspartame precursor, catalysed by thermolysin in aqueous and aqueous methanolic solutions was studied. Thermolysin with concentration as low as 10 M in 25% methanol can catalyse the synthetic reaction. The optimum methanol compositions at 4°C and 37°C were 50% and 25% respectively where an increase in peptide yield of 85% was obtained for both conditions as compared to that in water.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-aspartic acid - L-Phe-OMe L-phenylalanine methyl ester - N-cbz-L-Asp-Phe-OMe N-benzyloxycarbonyl-L-aspartyl-phenylalanine methyl ester All the % of methanol is a volume % in water unless otherwise specified.     

On the requirement of oxidizing reagent for the formation of a disulfide bond

Cyclization of a peptide through the formation of a disulfide bond between the SH groups of cysteines on the N- and C-terminals of peptide was studied in degassed water solution under vacuum. Cyclization went to completion although the solution was oxygen deficient (the number of oxygen molecules available for the reaction was at least 16 times less than the number of peptide molecules). This result indicates that, contrary to the common assumption, disulfide bond formation does not necessarily require an oxidant (O(2), I(2), etc.) to occur.     

Oxidation of amino acids and peptides in reaction with myeloperoxidase, chloride and hydrogen peroxide

Oxidation was studied of N-acetyl derivatives of cystine, cysteine, methionine and glycyltryptophan employing the myeloperoxidase-Cl--H2O2 system at pH 4.5, 6.0 and 7.0. Moreover, oxidation of pentapeptide composed of Leu-Trp-Met-Arg-Phe-COOH with myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and hypochlorite was also studied. It was found that amino-acid derivatives having an amino group bound to an acetyl residue react with functional groups of the side-chain. The -SH groups of N-acetylcysteine and the -SS- group of cystine oxidize to cysteic acid. Methionine residues oxidize to methionine sulphoxide, and tryptophan residues to a derivative of 2-oxoindolone. The same reaction products were obtained when respective amounts of hypochlorous acid were used instead of myeloperoxidase, Cl- and H2O2. Differences in the stoichiometry of reactions of myeloperoxidase-mediated oxidation and hypochlorite oxidation suggest differences in the reaction mechanisms of both studied systems. Interaction of the studied pentapeptide with myeloperoxidase-Cl(-)-H2O2 system as well as with hypochlorite showed that in the peptide molecule individual amino acids oxidize consecutively according to their susceptibility to oxidation. No splitting of peptide bonds was observed. Therefore, a modified peptide with methionine sulphoxide and and oxidized tryptophan incorporated into the molecule was obtained.     

Cryoenzymology of staphylococcal beta-lactamase: trapping a serine-70-linked acyl-enzyme

Various cryosolvents were investigated for their suitability in cryoenzymological experiments with beta-lactamase from Staphylococcus aureus PC1. On the basis of the minimal effects on the catalytic and structural properties of the enzyme, ternary solvents containing ethylene glycol, methanol, and water were found most suitable. The interaction of beta-lactamase with a number of substrates was studied at subzero temperatures. In general, the reaction profiles were similar to those in aqueous solution at above-zero temperatures, with the exception of the slower rates. For cephalosporin substrates, such as PADAC, in which the 3'-substituent may leave to form a more stable form of the acyl-enzyme [Faraci, W., & Pratt, R. (1985) Biochemistry 24, 903-910], this intermediate could be readily stabilized at subzero temperatures. At -40 degrees C the slow rate of deacylation in the reaction with the chromophoric substrate 6 beta-[(furylacryloyl)amino]penicillanic acid permitted the acyl-enzyme to be stoichiometrically accumulated. This intermediate was then stabilized at low pH with trifluoroacetic acid. Isolation by centrifugal gel filtration, followed by pepsin digestion, gave a penicilloyl-labeled peptide which was isolated by HPLC. Subsequent trypsinolysis of this peptide gave a single labeled peptide, corresponding to the octapeptide surrounding the active-site serine, Ser-70.     

A proton nuclear magnetic resonance study of gamma-glutamyl-amino acid cyclotransferase in human erythrocytes

Spin-echo NMR spectroscopy was shown to be a reliable technique for the monitoring of the in situ cleavage of gamma-Glu-Ala by gamma-glutamyl-amino acid cyclotransferase in whole erythrocytes and hemolysates. Of particular importance was the difference in chemical shifts between peptide resonances and those of the constituent amino acids. Using lysates of varying dilution, it was shown that the specific activity of the enzyme was not concentration-dependent, thus suggesting a lack of cytosolic low-molecular-weight-effectors or enzyme dissociation. Furthermore, the initial velocities of the reaction as a function of substrate concentration obeyed Michaelis-Menten kinetics with a Km = 2.0 +/- 0.3 mmol/l and Vmax = 137 +/- 7 mmol/h/l of cell water in 1H2O medium. Similar analysis in 2H2O medium revealed a solvent kinetic isotope effect of 1.9 +/- 0.4 at low substrate concentrations. The implications of this observation for the mechanism of the reaction are discussed. Cleavage of the peptide by a suspension of intact erythrocytes was at a rate 300 times less than the corresponding lysate flux, thus indicating the rate limitation by transport in the coupled system.