Deamidation and succinimide formation by gamma-N-methylasparagine: potential pitfalls of amino acid analysis

The biological function of the post-translationally methylated amino acid gamma-N-methylasparagine (gamma-NMA) in proteins is unknown. We are examining the premise that amide methylation protects against deamidation. The free amino acids Asn, gamma-NMA, Gln, and delta-N-methylglutamine (delta-NMG) were incubated at elevated temperature and a variety of pH conditions to assay for deamidation. Gln disappears 12- to 14-fold more rapidly than delta-NMG, and Asn hydrolyzes to Asp and NH3 as expected. However, the gamma-NMA deamidation rate is severely overestimated by simply measuring the disappearance of starting material because gamma-NMA undergoes a cyclization reaction in preference to deamidation. At pH 1 the predominant gamma-NMA reaction is formation of stable 3-amino-N-methylsuccinimide (NMS) and this occurs greater than 10-fold faster than Asn deamidation. At pH 4.0, 7.4, and 9.0 NMS is readily formed but it is unstable and partitions between the parent compound, gamma-NMA, and a second species, alpha-N-methylasparagine. At pH 7.4 and 9.0 gamma-NMA disappears 4-fold slower than Asn but the methyl amide hydrolysis rate is diminished by as much as 13-fold. The Asn incubations over the pH range 1-9 yield scant evidence of a succinimide intermediate. It is concluded that the amide methylation provides a unique reaction pathway and stabilization for the N-methylsuccinimide species. Amino acid analysis by o-phthalaldehyde postcolumn reaction fails to detect isoasparagine, alpha-N-methylasparagine, and NMS. Amino acid analysis by precolumn derivatization with phenyl isothiocyanate destroys NMS and therefore cannot quantitate this compound. The ninhydrin postcolumn derivatization method is able to detect and quantitate all of these amino acid species.     

Deamidation of the asparaginyl-glycyl sequence

The deamidation of Ac-Asn-Gly-NHMe and Ac-Isn-Gly-NHMe has been studied as a model for the facile deamidation of the Asn-Gly sequence in proteins. At alkaline pH, the product in each case is an identical mixture of Ac-alpha-Asp-Gly-NHMe (approximately 22%) and Ac-beta-Asp-Gly-NHMe (approximately 78%) as determined by n.m.r. spectroscopy. Because this same ratio is obtained from both Ac-Asn-Gly-NHMe and Ac-Isn-Gly-NHMe, the postulated mechanism, that deamidation proceeds through a cyclic imide intermediate, is confirmed. Unlike peptides of aspartyl esters, cyclization does not occur under nonaqueous conditions or at low pH in aqueous solution.     

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions have been studied in synthetic peptides to model these spontaneous processes that alter protein structure and function. We show here that the peptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala undergoes a rapid deamidation reaction with a half-life of only 1.4 days at 37 degrees C, pH 7.4, to give an aspartyl succinimide product. Under these conditions, the succinimide product can further react by hydrolysis (half-time, 2.3h) and by racemization (half-time, 19.5 h). The net product of the deamidation reaction is a mixture of L- and D-normal aspartyl and beta-transpeptidation (isoaspartyl) hexapeptides. Replacement of the asparagine residue by an aspartic acid residue results in a 34-fold decrease in the rate of succinimide formation. Significant racemization was found to accompany the deamidation and isomerization reactions, and most of this could be accounted for by the rapid racemization of the succinimide intermediate. Replacement of the glycyl residue in the asparagine-containing peptide with a bulky leucyl or prolyl residue results in a 33-50-fold decrease in the rate of degradation. Peptide cleavage products are observed when these Asn-Leu and Asn-Pro-containing peptides are incubated. Our studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively.     

Coupling of structural fluctuations to deamidation reaction in triosephosphate isomerase by Gaussian network model

We study the structural fluctuations of triosephosphate isomerase (TIM) by an elastic model, namely, the Gaussian network model (GNM), to identify a network of coupled motions in the allosteric communication between its deamidation and catalytic sites, and the promoting motions for the deamidation activity. For this, three TIM structures have been studied: one crystal structure and two model structures designed to describe different putative models for the deamidation reaction taking place at the subunit interface. The structural fluctuations have been mapped on the functional properties; then the differences in the fluctuations between the two models in relation to the deamidation reaction have been considered. The results demonstrate that the qualitative picture of the mean-square fluctuations and the correlations between the fluctuations are similar in both, but the differences may affect the observed barrier height of the deamidation reaction. The higher packing density at regions close to deamidation sites, reflected by the high-frequency fluctuating residues in the respective regions, the stronger positive correlation between the fluctuations of the deamidation sites, and enhanced positive correlation of the primary deamidation site with the extended vicinity of the catalytic region on the juxtaposed unit promote the probability of the deamidation reaction. The results in general emphasize the importance of structural fluctuations in enzyme reactions, as well as proposing the present methodology as a plausible approach for studies on the network of coupled promoting motions in protein functions.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Effect of viscosity on the deamidation rate of a model Asn-hexapeptide.

The effect of viscosity on the deamidation rate of a model Asn-containing hexapeptide (l-Val-l-Tyr-Pro-l-Asn-Gly-l-Ala) was assessed in aqueous solution and in solids containing varying amounts of poly(vinyl pyrrolidone) (PVP) and water. Stability studies were conducted at 0.1 mg/mL peptide and 0-50% PVP (w/w) in aqueous solution, and at 5% (w/w) peptide and different relative humidities (31.6, 53.1, 74.4 and 96%) in the solid state. The parent peptide and its deamidation products were analysed by reverse-phase high-performance liquid chromatography. Deamidation rates decreased with increasing solvent viscosity in a manner described by a semi-empirical mathematical model developed to describe this relationship. The results suggest that the motion of the Asn side-chain along the reaction coordinate is a function of the macroscopic solvent viscosity. However, the apparent energy barrier for the diffusive movement of the side-chain appears to be less than the energy barrier for that associated with macroscopic viscosity. The dependence of the deamidation rate on viscosity in both viscous solution and hydrated solids further demonstrates the importance of mobility in peptide deamidation.     

Influence of polymolecular events on inactivation behavior of xylose isomerase from Thermotoga neapolitana 5068

The inactivation behavior of the xylose isomerase from Thermotoga neapolitana (TN5068 XI) was examined for both the soluble and immobilized enzyme. Polymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95 degrees C, pH 7.0 and 7.9, the second phase was concentration-dependent. The enzyme was most stable at low enzyme concentrations, however, the second phase of inactivation was 3- to 30-fold slower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 XI revealed two distinct thermal transitions at 99 degrees and 109 degrees C. The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recoverable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivation was monophasic with a rate corresponding to the initial phase of inactivation for the soluble enzyme. The immobilized enzyme inactivation rate corresponded closely to the rate of ammonia release, presumably from deamidation of labile asparagine and/or glutamine residues. A second, slower inactivation phase suggests the presence of an unfolding intermediate, which was not observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were involved. Formation of a reversible polymolecular aggregate capable of protecting the soluble enzyme from irreversible deactivation appears to be responsible for the second phase of inactivation seen for the soluble enzyme. Whether this characteristic is common to other hyperthermophilic enzymes remains to be seen.     

Binding rates, O--S substitution effects, and the pH dependence of chymotrypsin reactions.

The pH dependence for acylation of alpha-chymotrypsin by N-acetyltryptophan p-nitrophenyl-, p-nitrothiophenyl-, ethyl-, and thiolethyl esters has been studied by the stopped-flow technique. Values for the acylation rate constant, k2, and the binding constant, KS, were obtained by using measurements of phenolate release, for the p-nitrophenyl esters, and proflavin displacement, for the ethyl esters. The oxygen esters tested have slightly higher k2 values, and substantially higher KS values relative to the analogous thiol esters. Whereas k2/KS for the thiolethyl ester is higher than that for the analogous oxygen ester, the k2/KS values for oxy- and thio-p-nitrophenyl esters are nearly identical. These data are interpreted to indicate rate-determining formation of a tetrahedral intermediate in acylation of alpha-chymotrypsin by p-nitrophenyl esters, and rate-determining breakdown of such an intermediate in the case of the ethyl esters. It is also concluded that the oxygen to sulfur substitution causes a substantial increase in the proportion of nonproductive binding in these substrates. pH dependent k2 and KS values were used to calculate values for k1 and k-1, the binding and debinding rate constants for the two p-nitrophenyl compounds. This is the first such calculation based on experimentally determined acylation rate constants.     

Mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase: kinetic studies

The catalytic mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) from family 39 of glycoside hydrolases has been subjected to a detailed kinetic investigation using a range of substrates. The enzyme exhibits a bell-shaped pH dependence of k(cat)/K(m), reflecting apparent pK(a) values of 4.1 and 6.8. The k(cat) and k(cat)/K(m) values for a series of aryl xylosides have been measured and used to construct two Br?nsted plots. The plot of log(k(cat)/K(m)) against the pK(a) of the leaving group reveals a significant correlation (beta(lg) = -0.97, r(2) = 0.94, n = 8), indicating that fission of the glycosidic bond is significantly advanced in the transition state leading to the formation of the xylosyl-enzyme intermediate. The large negative value of the slope indicates that there is relatively little proton donation to the glycosidic oxygen in the transition state. A biphasic, concave-downward plot of log(k(cat)) against pK(a) provides good evidence for a two-step double-displacement mechanism involving a glycosyl-enzyme intermediate. For activated leaving groups (pK(a) < 9), the breakdown of the xylosyl-enzyme intermediate is the rate-determining step, as indicated by the absence of any effect of the pK(a) of the leaving group on log(k(cat)) (beta(lg) approximately 0). However, a strong dependence of the first-order rate constant on the pK(a) value of relatively poor leaving groups (pK(a) > 9) suggests that the xylosylation step is rate-determining for these substrates. Support for the dexylosylation chemical step being rate-determining for activated substrates comes from nucleophilic competition experiments in which addition of dithiothreitol results in an increase in turnover rates. Normal secondary alpha-deuterium kinetic isotope effects ((alpha-D)(V) or (alpha-D)(V/K) = 1.08-1.10) for three different substrates of widely varying pK(a) value (5.15-9.95) have been measured and these reveal that the transition states leading to the formation and breakdown of the intermediate are similar and both steps involve rehybridization of C1 from sp(3) to sp(2). These results are consistent only with "exploded" transition states, in which the saccharide moiety bears considerable positive charge, and the intermediate is a covalent acylal-ester where C1 is sp(3) hybridized.     

Study of the photocycle and charge motions of the bacteriorhodopsin mutant D96N.

Absorption kinetic and electric measurements were performed on oriented purple membranes of D96N bacteriorhodopsin mutant embedded in polyacrylamide gel and the kinetic parameters of the photointermediates determined. The rate constants, obtained from fits to time-dependent concentrations, were used to calculate the relative electrogenicity of the intermediates. The signals were analyzed on the basis of different photocycle models. The preferred model is the sequential one with reversible reaction. To improve the quality of the fits the necessity of introducing a second L intermediate arose. We also attempted to interpret our data in the view of reversible reactions containing two parallel photocycles, but the pH dependencies of the rate constants and electrogenicities favored the model containing sequential reversible transitions. A fast equilibrium for the L2<==>M1 transition and a strong pH dependence of the M2 electrogenicity was found, indicating that the M1 to M2 transition involves complex charge motions, as is expected in a conformational change of the protein.     

Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.     

Kinetics of cyanide binding to Chromatium vinosum ferricytochrome c'

The kinetics of the reversible binding of cyanide by the ferric cytochrome c' from Chromatium vinosum have been studied over the pH range 6.9-9.6. The reaction is extremely slow at neutral pH compared to the reactions of other high-spin ferric heme proteins with cyanide. The observed bimolecular rate constant at pH 7.0 is 2.25 X 10(-3) M-1 s-1, which is approximately 10(7)-fold slower than that for peroxidases, approximately 10(5)-fold slower than those for hemoglobin and myoglobin, and approximately 10(2)-fold to approximately 10(3)-fold slower than that recently reported for the Glycera dibranchiata hemoglobin, which has anomalously slow cyanide rate constants of 4.91 X 10(-1), 3.02 X 10(-1), and 1.82 M-1 s-1 for components II, III, and IV, respectively [Mintorovitch, J., & Satterlee, J. D. (1988) Biochemistry 27, 8045-8050; Mintorovitch, J., Van Pelt, D., & Satterlee, J. D. (1989) Biochemistry 28, 6099-6104]. The unusual ligand binding property of this cytochrome c' is proposed to be associated with a severely hindered heme coordination site. Cyanide binding is also characterized by a nonlinear cyanide concentration dependence of the observed rate constant at higher pH values, which is interpreted as involving a change in the rate-determining step associated with the formation of an intermediate complex between the cytochrome c' and cyanide prior to coordination. The pH dependence of both the binding constant for the formation of the intermediate complex and the association rate constant for the subsequent coordination to the heme can be attributed to the ionization of HCN, where cyanide ion binding is the predominant process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of solution polarity and viscosity on peptide deamidation.

Deamidation kinetics were measured for a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0-50% w/w) and poly(vinyl pyrrolidone) (PVP, 0-20% w/w) at 37 degrees C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo-first order deamidation rate constants, k(obs), decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities >13 cp. Values of k(obs) also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side-chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k(obs). Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.     

Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides

The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects.     

Sequence and structure determinants of the nonenzymatic deamidation of asparagine and glutamine residues in proteins.

The rates of deamidation of Asn and Gln residues in peptides and proteins depend upon both the identity of other nearby amino acid residues, some of which can catalyze the deamidation reaction of the Asn and Gln side chains, and upon polypeptide conformation. Proximal amino acids can be contiguous in sequence or brought close to Asn or Gln side chains by higher order structure of the protein. Local polypeptide conformation can stabilize the oxyanion transition state of the deamidation reaction and also enable deamidation through the beta-aspartyl shift mechanism. In this paper, the environments of Asn and Gln residues in known protein structures are examined to determine the configuration and identity of groups which participate in deamidation reactions. Sequence information is also analyzed and shown to support evolutionary selection against the occurrence of certain potentially catalytic amino acids adjacent to Asn and Gln in proteins. This negative selection supports a functional role for deamidation in those non-mutant proteins in which it occurs.     

Reactivity toward deamidation of asparagine residues in beta-turn structures.

Mimetics of beta-turn structures in proteins have been used to calibrate the relative reactivities toward deamidation of asparagine residues in the two central positions of a beta-turn and in a random coil. N-Acetyl-Asn-Gly-6-aminocaproic acid, an acyclic analog of a beta-turn mimic undergoes deamidation of the asparaginyl residue through a succinimide intermediate to generate N-acetyl-Asp-N-Gly-6-aminocaproic acid (6-aminocaproic acid, hereafter Aca) and N-acetyl-L-iso-aspartyl (isoAsp)-Gly-Aca (pH 8.8, 37 degrees C) approximately 3-fold faster than does the cyclic beta-turn mimic cyclo-[L-Asn-Gly-Aca] with asparagine at position 2 of the beta-turn. The latter compound, in turn, undergoes deamidation approximately 30-fold faster than its positional isomer cyclo-[Gly-Asn-Aca] with asparagine at position 3 of the beta-turn. Both cyclic peptides assume predominantly beta-turn structures in solution, as demonstrated by NMR and circular dichroism characterization. The open-chain compound and its isomer N-acetyl-Gly-Asn-Aca assume predominantly random coil structures. The latter isomer undergoes deamidation 2-fold slower than the former. Thus the order of reactivity toward deamidation is: asparagine in a random coil approximately 3x(asparagine) in position 2 of a beta-turn approximately 30x (asparagine) in position 3 of a beta-turn.     

Human antibody Fc deamidation in vivo

Protein and peptide deamidation occurs spontaneously in vitro under relatively mild conditions. For antibodies and other therapeutic proteins, great effort is placed in manufacturing and storage to minimize this form of degradation. Concern has been especially great in cases where deamidation has been shown to impact protein activity. Here we monitored asparagine deamidation from a recombinant human antibody in humans and found that among the conserved sites, only Asn 384 deamidated at an appreciable rate. Under physiological temperature and pH conditions, in vitro antibody deamidation followed similar kinetics, indicating that simple incubation reactions may be used to model in vivo behavior. Endogenous IgG isolated from human serum possessed 23% deamidation at this site, further demonstrating that this modification is naturally occurring. Thus, deamidation generated in manufacturing and storage does not fully determine the patient exposure to the attribute. Instead, pharmacokinetic data along with the deamidation kinetics are combined to predict patient exposure. The deamidation rate can also be used to estimate the serum lifetime of antibodies. This approach could potentially be used to estimate turnover for other cellular or extracellular proteins.     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

The rate-determining step in pepsin-catalysed reactions, and evidence against an acyl-enzyme intermediate

To delineate further the pathway of pepsin-catalysed reactions, three types of experiments were performed: (a) the enzyme-catalysed hydrolysis of a number of di- and tri-peptide substrates was studied with a view to observing the rate-determining breakdown of a common intermediate; (b) the interaction of pepsin with several possible substrates for which ;burst' kinetics might be expected was investigated; (c) attempts were made to trap a possible acyl-enzyme intermediate with [(14)C]methanol in both a hydrolytic reaction (with N-acetyl-l-phenylalanyl-l-phenylalanylglycine) and in a ;virtual' reaction (with N-acetyl-l-phenylalanine) under conditions where extensive hydrolysis or (18)O exchange is known to occur. It is concluded that (i) intermediates in pepsin-catalysed reactions (aside from the Michaelis complex) occur subsequently to the rate-determining transition state, and (ii) an acyl-enzyme intermediate, if such is formed, cannot be trapped with [(14)C]methanol in these systems.     

Modeling the deamidation of asparagine residues via succinimide intermediates

Density functional theory (B3LYP/6-31G*) has been used to study the cyclization, deamidation and hydrolysis reactions of a model peptide. Single point energy calculations with the polarized continuum model drastically lower the activation energy for cyclization in a basic medium. Confirmation of the experimental results that cyclization is slower than deamidation in acidic media and the opposite is true in basic media has enabled us to propose mechanisms for both processes.