Peptide and Protein Carboxyl-terminal Labeling through Carboxypeptidase Y-catalyzed

A survey of carboxypeptidase Y-catalyzed carboxylterminal modification of short peptides in the presence of various amino acids revealed that transpeptidation occurred in significant yield only with peptides containing a proline at the penultimate or antepenultimate position. For these peptides, transpeptidation was shown to occur specifically at the carboxyl side of the proline, thus suggesting a determining role of this residue for transpeptidation. Two model peptides, YPFPGPI and YPFVEPI, were studied in detail. Initial yields of transpeptidation in the presence of various nucleophiles were compared. Among natural amino acids, the highest yield was obtained with methionine, followed by other amino acids bearing hydrophobic side chains. In order to transpose the method of transpeptidation to a protein, a variant of Escherichia coli methionyltRNA synthetase bearing the carboxyl-terminal GluPro-Met sequence was genetically created. Under the conditions optimized for the transpeptidation of YPFVEPI with methionine, this protein could be labeled specifically at its carboxyl-terminal end. Moreover, the parameters of the labeling reaction were in agreement with those observed in the transpeptidation of the model peptide.

Isotopically labeled polypeptides may be of considerable interest in various fields: protein purification and biochemical characterization, NMR, and physiological studies (2). A gap in the methods of investigation of biochemistry lies, however, in the difficulty of labeling polypeptides specifically at an extremi ty, as is possible, and very fruitful, with nucleic acids (3).
Chemical reagents are widely used to perform group-specific (4) or site-specific (5) modifications of proteins. However, in order to develop an amino-or carboxyl-terminal labeling process, the use of chemical reagents is problematic because they lack the required specificity, modifying also lysine side chains in one case, aspartic acid and glutamic acid side chains in the other case. This problem has been overcome by Jay (6) and by Jue and Doolittle (7), who designed a procedure for the specific labeling of the amino terminus of polypeptides. This method yielded some valuable information about the primary structure of the labeled protein, but other applications are limited by the fact that the conditions required by * This work was supported in part by Rhbne-Poulenc Sant& The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduert&ement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed.
the labeling procedure lead to the reduction or abolishment of protein activity. Therefore, an enzymatic approach, which was expected to preserve the native structure of the protein to be labeled, has been envisaged. It is based on the specificity of carboxypeptidases and on the ability of some of these enzymes to catalyze transpeptidations, i.e. to replace the carboxyl-terminal amino acid of a polypeptide with an exogenous nucleophile. Among this class of enzymes, CPD-Y,l a commercially available enzyme isolated from yeast, was especially suited, because its enzymatic properties (8-12), particularly its transpeptidase activity (13-17), have been extensively studied. Available data concerning CPD-Y-catalyzed transpeptidations deal, however, essentially with small unnatural substrates (N-blocked amino acids and dipeptides, mostly with an esterified or amidated carboxyl group) and with extremely high concentrations of the nucleophile.
Recently, CPD-Y was used to perform the labeling of @casein (18). Surprisingly, transpeptidation did not occur at the carboxyl-terminal position of the native protein, but occurred only after CPD-Y had removed the last 3 residues. This result, as well as preliminary attempts in our laboratory to transpose this method to other proteins, suggested that CPD-Y -catalyzed transpeptidation would be dependent on the carboxyl-terminal sequence of the substrate. One of our aims was therefore to elucidate the sequence determinants favoring transpeptidation. We also endeavored to design optimal conditions, particularly with respect to the nucleophile concentration, to maximize the specific radioactivity incorporated into the product and to minimize the amount of radioactive agent consumed in the labeling reaction. First, optimal conditions were defined using synthetic oligopeptides. Second, the results obtained on short natural substrates were extrapolated to a protein. However, to favor transpeptidation, the carboxyl-terminal extremity of the selected protein, Escherichiu coli methionyl-tRNA synthetase, was changed by genetic modification. We show here how this remodelling of the sequence of methionyl-tRNA synthetase allowed its specific labeling at the carboxyl-terminal end. ' The yield of labeling r, i.e. the ratio of the concentration of the labeled product P-p to the initial substrate concentration, is obtained from the solution of the differential equations relative to Scheme III.
Variation of r as a function of time yields a bell-shaped curve; the maximal value (r,,,ax) is given hy the following equation.
If we now suppose that the total rate of processing of the substrate P-X, represented by the A + A' constant, is independent of the nucleophile concentration [X*], we obtain the following expression for r, where a is a time constant directly proportional to [E]. The specific radioactivity, s, of the substrate at time t (s = [P-X*] /[P-q,,) and that, g, of the nucleophile supposed constant (if [P-XI a [X*], the isotopic dilution with released X is negligible) are related as follows.
Another important feature is the homogeneity, h, of the labeled product at time t, i.e. the ratio of the concentration of P-X (including P-X*) to that of the total protein in the assay, h = em z*t K + ,.x*1 .

G3)
Mo@ic&ion of Uze Gene of MethionyLtRNA Synthetase-Oligonucleotides preparation and site-directed mutagenesis were performed as described previously (25 ) and subjected to three precycles before the sample was loaded. PTH-derivatives were identified as described previously (28).

Transpeptidation of Short Peptides
In this part of the work, peptide substrates yielding transpeptidation in the presence of millimolar concentrations of nucleophiles were selected. Various nucleophiles were then compared, and the influence of experimental conditions (pH and temperature) was investigated using the selected peptides as model substrates. Finally, optimized conditions were employed in order to compare the transpeptidation yields obtained with various substrates.

Selection of Favorable Substrates for Transpeptidation-
The labeling of /3-casein (carboxyl-terminal sequence FPIIV) by Carles et ul. (18) indicated that, after the removal of the 3 carboxyl-terminal residues, CPD-Y became capable of ensuring transpeptidation provided a Pro was positioned in the S1 site. For this reason we brought particular attention in selecting model substrates of CPD-Y, to peptides bearing a penultimate Pro residue.
Peptide species that were not observed in the absence of the nucleophile were systematically searched for on chromatograms.
No new peptide was ever observed using LWMRFA as substrate. With RPPGFSPFR as substrate, the presence of a nucleophile 2 generated besides RPPGFSPF and RPPGFSP, a peptide which was found, by amino acid composition, to be RPPGFSPZ No trace of RPPGFSPFZ was detected. Since the substrate of transpeptidation was not the initial substrate but was the product RPPGFSPF of the f'irst peptidasic cycle, the peptide RPPGFSPFR was not further studied.
In the case of YPFPGPI, a new peptide appeared concomitantly with the hydrolysis product YPFPGP.
The relative proportion of the two products varied with the concentration of Z. Amino acid composition confirmed that the transpeptidation product was YPFPGPZ The peptide YPFPGPI was chosen as model substrate for further studies for two main reasons; (i) it is readily subject to transpeptidation (without prior hydrolysis) using relatively low (1 mM) concentrations of various nucleophiles, and (ii) the substrate and all the expected products carry an amino-terminal Tyr as common chromophore, thus facilitating the quantification of the reactions by absorbance or fluorescence detection. A peptide of close sequence, YPFVEPI, was also selected as a model substrate for similar reasons.
Test of Nucleophiles-The incorporation of the 20 common amino acids (except Ile), three amino acid amides, and norleucine (Nle) by transpeptidation with CPD-Y was tested on the substrate YPFPGPI.
The occurrence of transpeptidation was indicated by the appearance of a peptide with a chromatographic retention time different from that of the normal hydrolysis products. In the case of Ala, Val, Val-NHz, Met, Leu, Nle, Phe, Phe-NH*, Tyr, Thr, and Arg, the presumed transpeptidation product was submitted to amino acid analysis, verifying unambiguously the composition ProsTyrlPhel GlylZ1, where Z was the nucleophile involved in the assay. In the presence of Trp, a peptide appeared which contained a Trp residue, as indicated by its fluorimetric signal (AeX = 288 run, Lm = 348 nm), and was thus assigned to the transpeptidation product. In the cases of Cys, Gln, Lys, Asn, Ser, and His, plasma desorption mass spectrometry analysis confirmed that the new peak which appeared was due to the expected transpeptidation product. No new peptide could be evidenced in the remaining cases (Gly, Gly-NHz, Asp, Glu, Pro). However, in these cases, the transpeptidation product may have coeluted with one of the products obtained in the absence of added nucleophile.
To exclude this possibility, the peaks as- signed to YPFPGPI, YPFPGP, and YPFPG were further analyzed by mass spectrometry.
No trace of YPFPGPZ could be detected in these cases. Fig. 1 shows the initial transpeptidation yields observed at 37 OC and pH 6.5 for each studied nucleophile (2 or 20 mM). Higher yields are obtained with hydrophobic amino acids, the optimal side chains being those of Nle and Met. The influence of using a nucleophile whose carboxyl group is substituted for an amide group is not clear; it improves the yield obtained with Phe, but not with Val. Several transpeptidation constants (K) were determined, using either YPFPGPI or YPFVEPI as substrate (Table I). It is noteworthy that transpeptidation occurs more readily with YPFVEPI than with YPFPGPI; the transpeptidation constants with all nucleophiles are systematically 2-to 5-fold lower with YPFVEPI.
Effects of pH and Temperature-The transpeptidation constants K for the nucleophile Met, measured with YPFPGPI, at pH values ranging from 4.5 to 8.5, are plotted in Fig. 2. The K value shows little variation in the pH range 6.5-8.5, but is slightly increased under acidic conditions (pH 4.5-5.5). The same effect on K is obtained with Val as nucleophile and the peptide YPFVEPI as substrate (Table II). However, it must be pointed out that, in both cases, the reaction rate decreases considerably with increasing pH, limiting investigation to the pH 4.5-8.5 range for the peptide YPFPGPI and to the pH 4.5-6.5 range for the more anionic substrate YPFVEPI.
Contrary to the cases of Met and Val, the transpeptidation constant using Val-NH2 (YPFPGPI as substrate) shows a sharp decrease when the pH is raised from 6.5 to 8.5. This feature, which is consistent with observations by others (17), will be discussed later.
Finally, the favorable effect of lowering the temperature (pH 6.5, YPFVEPI as substrate) is illustrated by Fig. 3. Using Met as nucleophile, the K constant decreases Z-fold upon lowering the temperature of the assay from 50 to 4 "C.

Influence of the Carboxyl-terminal
Residue on Transpeptidation-The influence of the amino acid occupying the P'l position (at the carboxyl side of the scissile bound, i.e. the carboxyl-terminal position) was investigated by transpeptidation on substrates differing from YPFPGPI only by the carboxyl-terminal residue: YPFPGPM and YPFPGPR. Transpeptidation of YPFPGPR with 1 rnM Met was evidenced by following the appearance of the two products YPFPGP and YPFPGPM (Fig. 4). From this experiment, an initial yield of transpeptidation of 50% can be deduced, indicating a transpeptidation constant K very similar to that obtained with YPFPGPI as substrate (Table I). Using the nucleophile Phe, the transpeptidation constant with YPFPGPM (Table I) is identical to that obtained with YPFPGPI.
These results were expected, as the competition between water and the nucleophile requires the prior dissocia- tion of the cleaved amino acid from the acyl-enzyme; therefore, the K value, which reflects this competition, should not depend on the nature of the P'l residue.
It is noteworthy that, although the K constants for the Met nucleophile using the substrates YPFPGPR and YPFPGPI are almost identical, the reached maximal percentages of the YPFPGPM product are different. The maximal percentages observed are 2.5 and 25% with the substrates YPFPGPR and YPFPGPI, respectively. This discrepancy is due to the fact that the carboxyl-terminal P-R bond in YPFPGPR is hydrolyzed 30-fold more slowly than the P-I bond in YPFPGPI. Consequently, although the YPFPGPM product is formed in both cases at the same velocity from the acyl-enzyme, it does not accumulate in the assay with YPFPGPR, because it is further hydrolyzed by CPD-Y much more quickly than is the initial substrate YPFPGPR.
Influence Conditions are identical to those listed in the legend to Table II. ration of t3H]Met, glucagon was the only one which did not contain a Pro residue and nevertheless showed a significant yield of incorporation (Fig. 5B). In order to determine the position in the sequence where transpeptidation occurred, glucagon was submitted to transpeptidation in the presence of [3H]Met under the conditions described above. The reaction products at various times were analyzed by RPLC (ll-33% acetonitrile gradient, detection at 280 nm), and the resulting fractions were counted by liquid scintillation.
The radioactivity was shown to be associated with a single absorbance peak which was not observed in a control experiment performed in the absence of Met. The corresponding peptide species (I.5 nmol) was analyzed by plasma desorption mass spectrometry.
The measured mass (2593.3 ? 1 for the (M + H)+ ion species) corresponded to a 22-residue polypeptide, consisting in the l-21 fragment of glucagon, to which a carboxyl-terminal Met was added (calculated mass M = 2592.1). The resulting carboxyl-terminal sequence of this peptide was thus -AQDM. Two unlabeled species isolated by RPLC were characterized by mass spectrometry ((M + H)+ = 2462 and 2609). They corresponded to the l-21 (-AQD) and l-22 (-AQDF) fragments of glucagon, respectively. Noteworthy, no intermediate species longer than 22 residues could be detected on the chromatograms, which indicates that the residues 23-28, which are all hydrophobic, were cleaved very quickly by CPD-Y, as compared with the carboxyl-terminal Thr-29 residue.
The l-22 (-AQDF) fragment was prepared by a 30-min incubation of glucagon in the presence of 230 nM CPD-Y (37 C, pH 6.5) and was purified by RPLC. It was used as substrate to further determine the parameters of transpeptidation with Met. Contrary to the cases of all other substrates, when u + v'/v' was plotted as a function of l/ [Met] in order to determine the constant K, the intercept value of the straight line obtained was different from 1. The values given by linear regression were 1.3 k 0.1 mM for K and 6 k 0.3 for the intercept. This means that when the concentration of Met is increased, the initial yield of transpeptidation v'/v + v' reaches a limiting value of 0.17 & 0.01 instead of 1. The K constant, in this case, is the concentration of the nucleophile for which the yield equals half of this limiting value.
The fact that the limiting value of the initial yield of transpeptidation is lower than 1 reveals that the acyl-enzyme involving the l-21 fragment of glucagon can be hydrolyzed even though the Met nucleophile is bound to the S'l site of CPD-Y. This possibility, which was excluded from the mechanistic scheme (II), should thus be considered in some cases, depending on the substrate.

Labeling of a Protein
Having established the determining role of a Pro residue in the carboxyl-terminal sequence, the use of mutagenesis ap-  Table I. Buffer was 150 mM potassium phosphate (pH 6.5-8.5) or 50 mM potassium acetate (pH 4.5-5.5) and 1 mM EDTA. At pH 6.5, with YPFPGPI and Met, the same results were obtained with both buffers. CPD-Y concentrations insuring l%/min consumption of the substrate were: pH 4.5, 1.5 UM (YPFVEPI) or 12 nM (YPFPGPI); pH 5.5, 1.6 nM (YPFVEPI) or 6 nM (YPFPGPI); pH 6.5, 14 nM (YPFVEPI and YPFPGPI); pH 7.5,60 nM; pH 8.5,400 nM. ND, not determined. (19 nM CPD-Y, pH 6.5) was followed until 15% of the substrate was consumed. 5% was consumed in 75 min (2 "C), 7.5 min (25 "C), 3.4 min (37 "C), or 2.2 min (50 "C). the Glu-Pro-Met sequence. This sequence was chosen because the Pi and PP residues of the modified protein would be identical to those of the peptide YPFVEPI, the best identified substrate for transpeptidation. In addition, as the labeling would be performed with Met, one of the nucleophiles giving the highest yields of transpeptidation, the presence of a carboxyl-terminal Met would increase the carboxyl-terminal homogeneity of the labeled protein.
Incorporation of PHIMet in the Variant M556EPM Protein-The kinetics of transpeptidation of M547 and M556EPM in the presence of 2 pM [3H]Met (73 Ci/mmol) were followed by trichloroacetic acid precipitation (Fig. 6B). No incorporation of radioactivity was observed with the M547 variant of methionyl-tRNA synthetase. In contrast, with the M556EPM substrate, trichloroacetic acid-precipitable radioactivity increased as a function of time to a maximal value. The time required to reach this r,,,.. value can be shown to depend neither on [X] nor on the K constant, provided [x*1 << K. From this time, it could thus be deduced that the rate of hydrolysis by CPD-Y was about 40-fold lower for the protein substrate M556EPM than for the peptide YPFVEPM (both substrates were compared at a 20 pM concentration). This may be explained by a reduced accessibility to CPD-Y of the carboxyl-terminus of a folded protein as compared with that of a short peptide.
The presence of a denaturing agent (0.05% SDS) was shown to increase the rate of hydrolysis of the protein by a factor of at least 5, probably by partially unfolding the substrate. In the case of the protein substrate M547, it was even shown, by analysis of the released amino acids, that the presence of 0.05% SDS was necessary for any hydrolysis to occur. Incorporation of [3H]Met was thus tested as above, with both protein substrates, but now in the presence of 0.05% SDS. The same optimum level was obtained with the M556EPM variant, whereas again no significant incorporation could be evidenced with M547 (not shown).
In order to determine the K constant for the transpeptidation of M556EPM with Met, concentrations of this nucleophile in the range 0.1-l mM were employed (Fig. 6A). Initial rates of incorporation u' were calculated and a transpeptida- A, M556EPM (8.5 pM in 50 ~1) was incubated at 37 'C in the presence of 3 MM [3H]Met (73 Ci/mmol), plus 0.1 mM (IXl), 0.2 mM (+), 0.5 mM (m), or 1 mM (0) cold methionine, in buffer containing 50 mM potassium phosphate (pH 6.5), 10 mM 2-mercaptoethanol, 1 mM EDTA, 0.05% SDS, and 0.9 FM CPD-Y. Reactions were followed by trichloroacetic acid precipitation of 3-~1 aliquots. The yield of labeling is calculated as the ratio between the specific radioactivity of the total protein and that of [3H]Met. B, a control experiment was performed under the conditions described for A in the presence of 3 PM 13H]Met alone. The case of M556EPM (m) is compared with that of nonmodified methionyl-tRNA synthetase, M547 (ti).

19557
This value is close to the corresponding value for the peptide YPFVEPI (0.31 mM, Table II). It should, however, be kept in mind that the above determination of K with M556EPM as substrate is based on the hypothesis that v + v' is independent of Met concentration.
This condition was found to be true using YPFVEPI as substrate under similar conditions, at least up to 3 mM Met (not shown). From a mechanistic point of view (Scheme II), this could reflect that either (i) acylation is the limiting step of the reaction or (ii) the protein substrate concentration is far below its K,,, value. With the M556EPM substrate (which carries the same Pi and Pz residues as YPFVEPI), criterion (i) is expected to be conserved and the value of K,,,, on which depends (ii), is likely to increase, because of the reduced accessibility of the carboxyl terminus to CPD-Y. were trichloroacetic acid-precipitated (see "Experimental Procedures") during the course of the experiment in order to follow the reaction. After 2 h, the maximal level of incorporation (210,000 dpm) was reached and the reaction was stopped by the addition of diisopropylfluorophosphate (0.3 mM final concentration) followed by a 15-min incubation at room temperature.
No significant loss of activity of the synthetase occurred during this step, as determined by both ATPmPPi isotopic exchange (27) and tRNA aminoacylation (29) assays. The labeled protein was then added to the same quantity of unlabeled protein (2 mg) and dialyzed for 4 days at 4 "C against buffer containing 10 mM KZHPO., (pH 6.5) and 10 mM 2-mercaptoethanol.
It was determined by amino acid analysis that the amount of free Met remaining after dialysis was negligible.
Direct counting of the protein then allowed the measurement of the final specific radioactivity: 3,700 dpm/pmol. Thus, the reached yield of labeling, r (see "Experimental Procedures"), before isotopic dilution of the protein, was 4.6%. This yield is in reasonable agreement with the 6.7% value predicted from the experimental conditions and the model peptide YPFVEPI.
In preliminary experiments (not shown), smaller amounts of protein (cl0 pM in incubation) were labeled with a much better correlation between the predicted and obtained yields. Thus the lower yield observed The separation was performed on a Merck Superspher Cl8 column (250 X 4 mm) at 42 'C, using a 0.9 ml/min flow rate and the following gradient of acetonitrile in 0.1% trifluoroacetic acid O-16% in 5 min and then 16-48% in 40 min. 10 ~1 of each fraction (0.4 min) were counted after dilution in Picofluor. No radioactive material was eluted after 35% acetonitrile.
at high protein concentration may be attributed to the complex formation between methionyl-tRNA synthetase and Met (Kdim = 50 jrM (27)), which might lower by 25% the concentration of free Met under the conditions of the preparative labeling experiment.
Reversal of the Labeling-The labeled M556EPM protein (5 KM) was incubated in buffer containing 10 mM KZHP04 (pH 6.5) and 1 mM EDTA, either alone or in the presence of CPD-Y (0.35 PM), with or without SDS (0.05%). The reactions were followed using trichloroacetic acid precipitation as described under "Experimental Procedures." The results showed that incubation of the protein with CPD-Y resulted in the removal of the radioactive label. After 2 h at 37 'C, only 8% (in the presence of 0.05% SDS) or 19% (no SDS) of the initial counts remained.
This confirmed that the labeling of the protein had been caused by a CPD-Y-catalyzed phenomenon. The label of the protein incubated alone was completely stable under the same conditions, even though CPD-Y remained present at a l/25 molar ratio to the labeled methionyl-tRNA synthetase. This demonstrated a posteriori the efficiency of the diisopropylfluorophosphate inactivation of the peptidase at the end of the preparative labeling step. Characterization of the Radiolabeled Protein-The specificity of CPD-Y for the C-terminal extremity of polypeptide chains was expected to ensure the specificity of the labeling of M556EPM. This was confirmed by isolation of the labeled peptides after a proteolytic digestion of the protein. A chymotryptic digest of labeled M556EPM was analyzed by RPLC (Fig. 7). Four peaks (I-IV) represented 75% of the injected radioactive material. The remaining 25% was scattered in several small peaks, each accounting for less than 3% of the total. Noteworthy, with all nucleophiles used, the initial yield tends toward 1 when the concentration of the nucleophile rises. It allows one to describe transpeptidation by the single K value and confirms the mechanistic scheme (II) (see "Experimental Procedures"), presuming that no significant hydrolysis of the acyl-enzyme occurs when the S'i site is occupied by the nucleophile.
If this was not so, the maximal value of u//u + u' would strictly remain below 1. According to our model, the constant of transpeptidation should not depend on the leaving amino acid. This is well established in this study; the nature of the ultimate residue of the substrate can influence only the maximum yield of transpeptidation, through the ratio between the hydrolysis rates of P-X and P-Z. Using a given nucleophile 2, the better the substrate P-X, the higher the maximal yield obtained. According to the known specificity of CPD-Y (12), this means that better yields will be obtained if X is a hydrophobic residue rather than a small or charged residue.
On the contrary, Breddam et al. (14,17), working on Nblocked dipeptides, such as benzoyl-Ala-X, noticed that the yield of transpeptidation, using Leu-NH2 (0.25 M), Gly (1 M), or Gly-NH* (1 M) as nucleophiles, basically depended on the leaving amino acid X. When the side chain of X was hydrophobic, experimental yields were very low. This phenomenon was explained by the incomplete dissociation of the leaving amino acid during the deacylation step and by the presence of an independent binding site for a water molecule. Scheme II seems thus to be invalid in this case, probably reflecting the different nature of the used substrate.
Transpeptidation and Specificity of the Sfl Site of CPD-Y-All nucleophiles tested under identical assay conditions on the substrate YPFPGPI are sorted in Fig. 1 according to decreasing efficiency for transpeptidation.
It is obvious that transpeptidation works better with hydrophobic nucleophiles, especially with amino acids bearing a long aliphatic side chain and with Phe. Although the transpeptidation constant K may not be simply regarded as the dissociation constant for an acyl-enzyme:nucleophile complex, it partly reflects the interaction of the nucleophile with the S'i site of CPD-Y (the S'l site is the subsite of the active center which interacts with the carboxyl-terminal residue of the substrate). The observed preference for hydrophobic side chains is in agreement with the known specificity of this site (12).
This preference for hydrophobic side chains was not observed by Widmer et ul. (16) who worked on the substrate benzoyl-Ala-O-methyl.
However, these authors (16), using amino acids and amino acid amides as the nucleophile 2, compared the maximal percentages of benzoyl-Ala-2 formed rather than initial rates of product appearance. For achieving maximal yields of the transpeptidation product, they always used the highest possible concentration of the nucleophile, ranging from 0.15 to 3 M. Moreover, in their case, the substrate was an ester, and the reactions were carried out at pH 9.5.
Comparison of Amino Acids and Amino Acid Amides as Nucleophiles-From already mentioned studies, it appears that transpeptidations with amino acid amides differ from those carried out with amino acids; reactions with amino acid amides are generally much more efficient and depend little on the nature of the side chain (15,16). Transpeptidations with amides, unlike those performed with amino acids, were also shown to crucially depend on pH (17) in the pH 5-8 range. The transpeptidation of Cbz-Ala-Ala with Gly or Gly-NH2 (1 M) was compared at various pH values. With Gly, the yield remains at its maximal value between pH 5 and 8. With Gly-NHZ, the yield jumps from nearly 0% at pH 6 to nearly 100% at pH 8.
These differences between amino acids and amino acid amides as nucleophiles in CPD-Y-catalyzed transpeptidation remain unelucidated.
It has been proposed (13, 16) that the pKO of the a-amino group of the nucleophile, which is generally 1.5 units higher for an amino acid than for the corresponding amide, may play a role, although this property was not sufficient to explain satisfactorily the variations of the yield uersus pH (17). This explanation does not, however, take into account the fact that once the nucleophile is bound to CPD-Y, its amino group is surrounded by the extremely powerful charge relay network of the proteolytic enzyme, which may considerably lower the pKa value. To explain the discrepancies, it must thus be imagined that, as suggested by Breddam et al. (l7), amino acids and amino acid amides bind differently to the S'i site of CPD-Y. The distinct variations of transpeptidation towards pH may then reflect the ionization of several residues of the enzyme differently involved in the binding.
Our present results concerning transpeptidation of the peptide YPFPGPI with Met, Val, and Val-NH2 are consistent with the previously mentioned results. They further indicate that the variations with pH, specifically observed with amino acid amides, mostly reflect the ionization of a charged residue of the enzyme involved in the tixation of the nucleophile and thus could be expected to be similar for any substrate.
The Importance of a Penultimate Proline in optimizing Trunspeptidution-Discrepancies were outlined in the first paragraph of the discussion section between the mechanism of transpeptidations described in the literature and that established in this study using YPFPGPI and YPFVEPI as substrates. In addition, the smallest transpeptidation constant measured in this study (0.3 mM using Met and the peptide YPFVEPI) is 100-1000 times lower than the values extrapolated from the analysis of previous data by others. The only similar situation is that of peptides related to /3casein (18). However, as outlined already, such comparisons should be considered cautiously because of the different experimental approaches used. More reliable information is obtained from the result of the incorporation of tritiated Met in various peptide substrates