Báo cáo khoa học: Fragments of pro-peptide activate mature penicillin amidase of Alcaligenes faecalis pdf

Fragments of pro-peptide activate mature penicillin amidase of Alcaligenes faecalis Volker Kasche, Boris Galunsky and Zoya Ignatova Institute of Biotechnology II, Technical University Hamburg-Harburg, Hamburg, Germany Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (k cat /K m )forthehydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin ami- dase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca 2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addi- tion of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chainraisedtheactivityupto2.3-foldandincreasedthe specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number (k cat ). Keywords: Alcaligenes faecalis; pro-peptide; enzyme activa- tion; penicillin amidase; site-directed mutagenesis. Penicillin amidases (PA, EC 3.5.1.11) are biotechnologically important enzymes used in the production of semisynthetic b-lactam antibiotics. Penicillin amidases are present in a variety of organisms including bacteria, yeast and fungi, and they all diverge from a common evolutionary ancestor [1]. The physiological function of penicillin amidases in vivo is not yet known. It has been speculated that they are involved in the metabolism of aromatic compounds as carbon sources [2], as the pac gene is localized in the proximity of genes coding for enzymes involved in degradation of 4-hydroxyphenylacetic acid [3]. PA belongs to the structural superfamily, the Ntn (N-terminal nucleophile) hydrolases, in which all members are related in that the first event in the autocatalytic processing of the inactive precursor reveals a catalytic serine, threonine or cysteine at the N-terminal position [4]. The processing of the inactive PA precursor to mature periplas- mic enzyme has been studied in detail for the Escherichia coli enzyme. The nascent pac gene encodes a prepro-PA (97 kDa) containing an N-terminal signal peptide (pre- sequence, 26 amino acids) that is cleaved upon crossing the cytoplasmic membrane via the Tat pathway [5]. The crystal structures of E. coli PA [6], of Providencia rettgeri PA [7], as well as the mutant slow processing E. coli pro-PA [8] provide insight into the catalytic mechanism and clarify the role of the N-terminal serine of the B-chain as a single catalytic residue. The inactive pro-PA (92 kDa) is activated by multiple proteolytic cleavages starting with an intra- molecular autocatalytic step between Thr263 and Ser264, which generates the B-chain (62 kDa) [4,6,8,9]. The pro- peptide (known also as linker or spacer peptide, 54 amino acids) is further sequentially removed from the C-terminus of the A-chain in intra- and intermolecular processing events, resulting in a release of the A-chain (23 kDa) [8], found as a dominating form in the commercial PA preparations. While the presequence mediates translocation through the membrane, the function of the pro-region is still unknown. Even though such an exclusion mechanism of short peptides from inactive precursors in the maturation process is a widely spread in living systems, the exact role of the pro-domain is not completely understood. For some proteases such as subtilisin [10], nerve growth factor [11] and a-lytic protease [12], the pro-region is required for correct folding in vivo or refolding in vitro. Furthermore, the pro- domain accelerates the structure formation by facilitating formation of correct disulfide bonds [11]. Partial or whole deletions in the pro-sequence affect maturation and correct processing of nerve growth factor [13]. While Alcaligenes faecalis PA shares the lowest sequence homology to E. coli PA in the penicillin amidase family from the Gram-negative bacteria, the precursor organization resembles that of the E. coli PA, starting with an N-terminal presequence (26 amino acids), fol- lowed by the A-chain (202 amino acids), pro-region (37 amino acids), and B-chain (551 amino acids) [14]. As both enzymes possess the same substrate specificity and share extensive similarities in functionally important amino acid residues, it is expected that their molecular mechanisms of processing are similar, e.g. the pro-peptide is step-wise proteolytically removed in the maturation process yielding Correspondence to V. Kasche, Institute of Biotechnology II, Technical University Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany. Fax: + 49 40 42787 2127, Tel.: + 49 40 42878 3018, E-mail: kasche@tu-harburg.de Abbreviations: IEF, isoelectric focusing; NIPAB, 6-nitro-3-phenyl- acetamido benzoic acid; Ntn, N-terminal nucleophile; PA, penicillin amidase. Enzyme: penicillin amidase (EC 3.5.1.11). (Received 9 July 2003, revised 4 September 2003, accepted 6 October 2003) Eur. J. Biochem. 270, 4721–4728 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03871.x the mature two-chain enzyme [14,15]. Recently, we gave experimental evidence for the step-wise shortening of the pro-peptide of A. faecalis PA by the isolation of the last two active forms with different length of the A-chains [15]. Comparative studies of the E. coli and A. faecalis PA showed that the specific activity of A. faecalis enzyme in the cell homogenate is about fivefold higher. After purification to homogeneity only twofold higher specific activity of A. faecalis PA compared to E. coli PA was measured [15]. The difference in the specific activity of the A. faecalis PA in the homogenate and as a purified protein indicates that an activating compound is lost during the purification of this enzyme. This is verified in this study on wild-type A. faecalis PA, where we demon- strate that fragments from the pro-peptide act as activa- tors in vitro. Furthermore, our results show that inhibiting the later steps of the pro-peptide removal in vivo by introduction of specific point mutations in the pro-domain increased the specific activity of the mutant enzymes with extended A-chains. The observed higher specificity con- stants of the mutants for benzylpenicillin hydrolysis are mainly due to an increase in the turnover number (k cat ). Experimental procedures Bacterial strains, plasmid construction and growth conditions Plasmid pPAAF for the in vivo synthesis of A. faecalis prepro-PA was constructed as follows. A 2360 bp PCR fragment covering the region from 13 nucleotides upstream from the start codon of A. faecalis pac with the altered RBS and Shine–Dalgarno sequence was amplified using the following primers 5¢-CGAATTCTGAGGAGGTAGTAATGCAGAAAGG GCT-3¢ and 5¢-CCTCCAAGCTTAAGGCAGAGGCTG-3¢ (ARK- Scientific GmbH, Germany) with chromosomal DNA from A. faecalis ATCC 1908 as a template. The product was double digested with EcoRI and HindIII and cloned into the multiple cloning site of pMMB207 [16] yielding a pPAAF plasmid. The last was used as a template for the introduction of site-specific mutations (Table 1) into the pro-peptide coding sequence using QuickChange Mutagenesis Kit (Stratagene, the Netherlands). All mutations were verified by DNA sequen- cing (SeqLab, Germany). The A. faecalis pac gene was expressed under the tac- promoter and therefore induced by 0.5 m M isopropyl thio-b- D -galactoside. During all genetic manipulations the host cells E. coli DH5a were grown aerobically in Luria– Bertani medium supplemented with 25 lgÆmL )1 chloram- phenicol as a selection marker [17]. Transformed E. coli DH5a cells were plated on LB agar medium with a nitro- cellulose filter. Positive clones harboring the A. faecalis pac gene were screened phenotypically for PA-activity with the chromogenic substrate 6-nitro-3-phenylacetamido benzoic acid (NIPAB) [18]. Purification of wild-type A. faecalis penicillin amidase and its pro-peptide mutants For expression E. coli BL21(DE3) cells were transformed with either pPAAF or plasmids carrying mutations in the pro-peptide and were cultivated at 28 °C in minimal M9 medium, containing 2.5 gÆL )1 glucose. Six hours after induction with isopropyl thio-b- D -galactoside (0.5 m M )the cells were harvested by centrifugation at 1700 g for 15 min. Furthermore, they were fractionated into periplasmic and cytoplasmic fractions by cold mild osmotic shock procedure as described previously [19]. The wild-type A. faecalis PA and the pro-peptide mutants were purified from the concentrated supernatant by anion-exchange chromatography using the same proce- dure as described in [15,20]. All eluted protein fractions were desalted into 30 m M Tris buffer, pH 7.5, and concentrated using Amicon centrifugal filters (cut-off 10 kDa). The homogeneity of the enzyme forms was analyzed by isoelec- tric focusing (IEF) and SDS/PAGE [21]. In the IEF experiments ready for use ServalytÒPrecotesÒ 3–10 gels with supplied buffer systems (Serva, Germany) were run according to the instructions of the manufacturer on a Multiphor II (LKB Bromma, Sweden) apparatus. Assay for penicillin amidase activity and active site titration The PA activity was measured by a spectrophotometric assay with the chromogenic substrate NIPAB [20]. Under standard conditions (pH 7.5, 25 °C, 125 l M NIPAB), the specific activity is defined as a change in the absorbance at 380 nmÆmin )1 , per protein content expressed as an absorbance at 280 nm (DA 380 min )1 ÆA 280 )1 ). Pure E. coli PA with a concentration 1 mgÆmL )1 possesses an A 280 value of 2.0 [20]. The formula for recalculation of the acti- vity measured with the same substrate at 405 nm is: DA 405 min )1 ¼ 0.94 · DA 380 min )1 . The molar concentrations of the enzymes were determined by active site titration [22]. Equal amounts of wild-type Table 1. Amino acid substitutions in the pro-peptide generated by site-directed mutagenesis. The amino acids introduced by mutagenesis are shown in bold. The sequences of the mutated pro-peptides start from the N-terminus. Short assignment of the mutated pro-peptides Amino acid sequences of the pro-peptides Wild-type pro-sequence QAGTQDLAHVSSPVLATELERQDKHWGGRGPDFAPKA T206P QAGPQDLAHVSSPVLATELERQDKHWGGRGPDFAPKA T206G QAGGQDLAHVSSPVLATELERQDKHWGGRGPDFAPKA T206GS213G QAGGQDLAHVGSPVLATELERQDKHWGGRGPDFAPKA T206GS213GT219G QAGGQDLAHVGSPVLAGELERQDKHWGGRGPDFAPKA 4722 V. Kasche et al. (Eur. J. Biochem. 270) Ó FEBS 2003 A. faecalis PA or enzymes with point mutations in the pro- peptide were incubated with different amounts of phenyl- methanesulfonyl fluoride in phosphate buffer pH 7.5, I ¼ 0.2 M for 30 min. The residual activity was measured spectrophotometrically using NIPAB as a substrate. Determination of the kinetic parameters The PA-catalyzed hydrolysis of benzylpenicillin was per- formed at 25 °C and pH 7.5 (phosphate buffer I ¼ 0.2 M ). The used substrate concentrations were 5, 10, 20, 40, 60 and 80 l M . Enzyme concentration in the reaction mixture were between 3.2 · 10 )11 M and 10 · 10 )11 M . Periodically aliqu- ots were withdrawn and immediately analyzed by HPLC as described previously [23]. The initial rates (about 10% substrate exhausting) were determined on the basis of the increase of phenylacetic acid concentration as a function of time. Five to six points were measured. The initial rates were calculated by linear regression analysis using PLOTIT software, version 3.14 (Scientific Programming Interfaces, 1994). The initial rates at each substrate concentration were average values of three independent experiments. The values of the steady-state kinetic parameters K m and k cat for A. faecalis PA and pro-peptide mutants were calculated using reversed Eadie–Hofstee plots. Determination of the bound calcium ion The calcium ion content in the purified A. faecalis forms (protein concentration 1 mgÆmL )1 ) was measured by Induced Coupled Plasma-Atom Emission Spectroscopy (ICP-AE-spectrophotometer, Perkin-Elmer). In order to rule out any unspecific bound calcium ions, the purification was performed with calcium-free buffers and additionally before the measurement the purified proteins were trans- ferred into double distilled water with Bio-Rad HR 10/10 desalting column. Calcium ion content of the blank (double distilled water treated on the same way as the sample) was zero. In vitro influence of the pro-peptide and fragments of it on the activity of purified A. faecalis penicillin amidase The activation of A. faecalis PA in vitro was tested with chemically synthesized fragments of the pro-peptide (11- mer, 20-mer, 29-mer and the whole pro-peptide 37-mer; ARK-Scientific GmbH, Germany). The sequences of all oligopeptides were derived from the pro-peptide as presented in Table 2. Purified A. faecalis PA with an isoelectric point (pI) of 5.3 (15 n M ) was incubated for 15 min at 25 °C in phosphate buffer pH 7.5 I ¼ 0.2 M with the above oligopeptides in the concentration range 0–75 n M . Then the mixture was subjected to activity measurements using NIPAB as a substrate. Results and discussion Sequence alignment and comparison with E. coli penicillin amidase The A. faecalis PA shows 40% protein sequence identity with the E. coli PA (Fig. 1). Taking conservative substitu- tions into account, the homology rises above 48%. The key catalytic and oxyanion hole forming residues [24] (Ser264, Gln286, Ala332, Asn504, Asn505, Arg526; numbering is according to the amino acid sequence of E. coli pro-PA [25]) are strictly conserved in the A. faecalis PA (Fig. 1) and in the other members of the PA family [14,26]. Another interesting aspect of this comparison is that the most of the conserved clusters, e.g. residues 133–148, 284–316, 440–446, 490–507, and 739–751, are in the vicinity of the active site. While the enzymes of the PA family do not require a calcium ion as a cofactor, the crystal structures of E. coli PA (PDB access number 1PNK), of the slow processing Gly263Thr mutant E. coli pro-PA (PDB access number 1E3A), and of the P. rettgeri mutant Bro1 PA [7] reveal a tight bound calcium ion in the structure. ICP-AES analysis confirmed the presence of one calcium ion in the A. faecalis PA molecule. Five of the six calcium co-ordinating residues identifiedintheE. coli PA (Glu152, Asp336, Val338, Asp339, and Asp515) are fully conserved in the A. faecalis PA (Fig. 1). These residues are also conserved among the other PA members of the Enterobacteriaceae Kluyvera cytrophila and P. rettgeri (see the alignment published by Verhaert et al. [14]). The largest divergence exists in the pro-peptide removed during maturation. The crystal structure of mature E. coli PA reveals that both chains form a pyramid with the active site serine located at the base of a deep cone [6]. In the E. coli pro-PA the active site cleft is covered by the pro-peptide [8], localized on the surface of the pro-enzyme molecule and flanking the superficial C-terminal part of the A-chain and the deep concealed N-terminus of the B-chain. The pro- region of A. faecalis pro-PA is 17 amino acids shorter than the E. coli pro-PA. This deletion is localized in the first superficial part of the pro-peptide, in the loop before the a-helix structure. Loops as flexible structural elements easily tolerate deletions or insertion of extra residues without perturbation of the entire structure [27]. Until now, no direct evidence exists about all amino acids participating in the autocatalytic maturation process. Table 2. Amino acid sequences of the synthetic oligopeptides. The sequences of the synthetic oligopeptides correspond to the (fragment) sequence in the wild-type A. faecalis pro-peptide starting from the N-terminus. Length of the oligopeptide Amino acid sequence in a single letter code 11-mer QAGTQDLAHVS 20-mer QAGTQDLAHVSSPVLATELE 29-mer QAGTQDLAHVSSPVLATELERQDKHWGGR 37-mer QAGTQDLAHVSSPVLATELERQDKHWGGRGPDFAPKA Ó FEBS 2003 Penicillin amidase activation by pro-peptide fragments (Eur. J. Biochem. 270) 4723 Possible candidates, such as the N-terminal SN sequence (Ser264, Asn265) of the B-chain, and Gly284 [28], are fully conserved in both penicillin amidases (Fig. 1). Lys273, described as a residue responsible for a pH-dependent processing [29], is conservatively substituted in the A. fae- calis sequence with an arginine which provides the necessary sidechainwithabasicpK a . The catalytically active serine at the N-terminus of the B-chain being totally conserved (Fig. 1) reveals the necessary requirement for an efficient self-processing prerequisite for the PA activity [4]. These sequence considerations support the assumption for similar processing mechanism of both A. faecalis and E. coli PA. Moreover, our previous study with A. faecalis PA [15] supports with experimental evidence the assumption for a sequential removal of the pro-peptide from its C-terminus, similar to the maturation of E. coli PA [9]. In vitro influence of the pro-peptide and its fragments on the activity of A. faecalis PA The stable processed form of A. faecalis PA, expressed in E. coli, was produced and purified as already described [15]. Typically, the purified final mature form of the enzyme with a completely removed pro-peptide appeared homogeneous with respect to IEF and SDS/PAGE analysis with an isoelectric point (pI) of 5.3. The total activity, used to evaluate the purification yield showed a 57% loss after the first purification step, the concentration of the periplasmic fraction by ultrafiltration (molecular size cut-off 10 kDa) [15]. An addition of this filtrate to purified A. faecalis PA led to more than twofold increase of specific activity and 86% of the total activity was restored (data not shown). The pro- peptide (37 amino acids) is sequentially shortened during the maturation process and the resulting fragments, acting obviously as activators, are probably removed from the active enzyme in this step, remaining in the ultrafiltrate. This prompted us to investigate the possible influence of the whole pro-region or fragments of it with random lengths on the activity of the A. faecalis PA. The incubation of the chemically synthesized oligopeptides with purified A. fae- calis PA (pI 5.3) at different molecular ratios led to an activation of PA and the specific enzyme activity increased up to 2.3-fold (Fig. 2). The highest activation was measured for the shortest oligopeptide (11mer) with an activation effect being concentration dependent. Increasing the amount of the 29-mer over the stoichiometric ratio had hardly any significant effect. In the case of 11-mer oligopep- tide the activity raised up to a ratio 1 : 2 (PA/11-mer) Fig. 1. Amino acid sequence alignment of E. coli PA and A . faecalis PA. Identical residues are shadowed, similar substitutions are framed. Numbering is according to the amino acid sequence of the E. coli pro-PA [25] starting with the first amino acid of the A-chain. The signal peptide cleaved off after translocation is numbered in the opposite direction. d, catalytic residues and residues from the oxyanion hole; h,calciumion coordinating residues. The residues of the pro-peptides in both sequences are underlined. 4724 V. Kasche et al. (Eur. J. Biochem. 270) Ó FEBS 2003 (Fig. 2), although over the physiological ratio 1 : 1 the activity increased only with additional 20%. The non- covalent interactions between the shortest oligopeptide and PA seem to be dynamic and reverse, therefore concentra- tions over the stoichiometric ratio increase the number of oligopeptide bound to the enzyme resulting in a higher enzymatic activity. In the experiments with the oligopeptide with a length of the whole pro-peptide (37-mer), the PA activity measure- ments were problematic. During the first 10 s after mixing with the substrate NIPAB an increase of the absorbance at 380 nm was detected, followed by a phase where practically no absorbance change was observed, even when the substrate was not exhausted (data not shown). Most probably, during the preincubation of purified A. faecalis PA(pIof5.3)with the 37-mer oligopeptide (representing the whole pro-pep- tide), it fits once again into the entrance of the cone and covers the active site, which results in restricted diffusion of the substrate molecules to the catalytic serine. Effects of the inhibition of the complete proteolytic processing of the pro-peptide on the penicillin amidase activity In a previous study we succeeded in isolating the last two active forms of A. faecalis PA. ICP-AES analysis confirmed that both forms contained one tightly bound calcium ion leading to the conclusion that calcium ion binding precedes the processing of pro-PA. By mass-spectrometry analysis we showed that the observed higher molecular mass of the A-chain of the form with pI 5.5 compared to the A-chain of the last maturation form with pI 5.3, is due to the four amino acids from the pro-peptide still remaining covalently attached to the A-chain [15]. Therefore, the first position mutated was Thr206 and we exchanged it with Pro and Gly (Table 1). The resulting mutant PA-precursors were con- cisely assigned by the single letter code of the substituted amino acid and its position, followed by the code of the replacing amino acid. The numbering is according to the published primary structure of A. faecalis pro-PA [14], starting with the N-terminal amino acid of the A-chain. The measured specific activity of the T206P mutant was lower, being about 85% of the specific activity of the wild-type completely processed A. faecalis PA (pI 5.3) (Table 3). The T206P mutant appeared to undergo further normal pro- teolytic processing leading to a completely processed PA form with pI 5.3 (Fig. 3A, lane 3). Table 3. Specific activity of the wild-type A. faecalis PA (pI 5.3) and the site-directed mutants. Activity was measured with purified proteins. Each specific activity value is an average of three measurements. The k cat values for NIPAB hydrolysis were estimated from the specific activity and the active site titration data and were calculated to be: wild-type A. faecalis PA 82 s )1 (see also [15]), T206G mutant 131 s )1 , T206GS213G mutant 152 s )1 , T206GS213GT219G 185 s )1 . A. faecalis PA forms Specific activity DA 380 min )1 ÆA 280 )1 Wild-type (pI 5.3) 2.0 ± 0.1 T206G mutant 3.2 ± 0.2 T206P mutant 1.7 ± 0.1 T206GS213G mutant 3.7 ± 0.2 T206GS213GT219G mutant 4.5 ± 0.3 Fig. 3. Processing patterns of purified mutant A. faecalis PA precursors with alterations at positions 206, 213 and 219. (A) IEF stained with Coomassie blue, Lanes: M, isoelectic point marker; 1, purified last two maturation forms of the wild-type A. faecalis PA with pI 5.3 and 5.5; 2, T206G mutant; 3, T206P mutant. (B) SDS/PAGE stained with Coo- massie blue. Lanes: 1, purified last maturation form of the wild-type A. faecalis PA (pI 5.3); 2, T206GS213GT219G mutant; 3, T206GS213G mutant; 4, A. faecalis PA (pI 5.5); 5, T206G mutant. Fig. 2. In vitro influence of fragments of the pro-peptide on the A. fae- calis PA (pI 5.3) activity. The activity measurements were performed as described in Materials and methods with 15 n M enzyme and oligo- peptides in the concentration range 0–75 n M . The starting point is the activity of A. faecalis PA (pI 5.3) without oligopeptides, which was taken as 1. d, 11-mer; m,20-mer;s, 29-mer. Ó FEBS 2003 Penicillin amidase activation by pro-peptide fragments (Eur. J. Biochem. 270) 4725 Our previous mutational experiments showed that the replacement of the original Thr in the pro-sequence of E. coli pro-PA by Gly retards the rate of its processing which allowed isolation of the precursor [8,9]. Furthermore, the Thr206 was also mutated to Gly (T206G), which led to the predominating active form of PA with pI 5.5 (Fig. 3A, lane 2). The specific activity of T206G mutant was 60% higher compared to the wild-type A. faecalis PA with pI of 5.3 (Table 3). SDS/PAGE analysis under denaturing con- ditions gave a double band for the A-chain (Fig. 3B, lane 5). The lower band corresponds to the size of the completely processed A-chain of A. faecalis PA with pI 5.3 and the upper one (marked as A + P) is of the approximate size of the A-chain plus fragment of the pro-peptide. The further cleavage of the remaining four amino acids from the pro- peptide at 25 °C and pH 7.5 was a relatively slow process and even after 312 h incubation approximately 30% was not converted into the form with pI of 5.3 (Fig. 4A). The question arose, whether the extended length of the A-chain by four amino acids affects the catalytic or the binding properties of the enzyme. The steady-state kinetic parameters K m and k cat for benzylpenicillin hydrolysis are summarized in Fig. 5. Whereas the K m values for benzyl- penicillin hydrolysis by A. faecalis PA (pI 5.3) and T206G mutant were equal, the k cat value for T206G mutant was about 1.5-fold higher (Fig. 5). The similarity in the K m values was not surprising, as the remaining four amino acids from the pro-peptide cannot cover the entrance to the active site and therefore do not influence the substrate binding properties of the enzyme. Fragments of the pro-peptide still remaining covalently attached to the mature A. faecalis PA can probably influence the stabilization of the transition state of the rate limiting step (formation of the acyl-enzyme intermediate) thus leading to higher k cat values. A similar effect was observed for cephalosporin acylase from Pseu- domonas sp. 130 [30]. Although the replacement of T206 by Gly led to a retarded processing of the mutant precursor, the further removal of the pro-peptide could not be blocked completely. All purified samples of T206G contained traces of the completely processed PA with pI 5.3 (Fig. 3), therefore additional site-specific amino acid substitutions were intro- duced into the pro-peptide coding region of A. faecalis PA (Table 1). In the in vitro experiments with chemically synthesized oligopeptides the highest activation was observed with the 11-mer peptide (Fig. 2), thus the position of Ser213 was chosen for the next replacement. The processing of E. coli pro-PA starts with an intra- molecular autoproteolytic cleavage between Thr263 and Ser264 yielding the free N-terminal serine of the B-chain [6]. Detailed mapping of some of the further shortening of the pro-region revealed Asn241-Arg242 and Asp223-Arg224 to be the next cleavages in the maturation process [9]. The Asn241-Arg242 bond is within the a-helical region (resi- dues 240–251 [8]). The a-helix propensity analysis of the Fig. 4. Stability of purified mutant A. faecalis PA precursors monitored by IEF. (A) Purified T206G mutant dissolved in 1 m M Tris/HCl pH 7.5 was incubated at 25 °C for 24 h (lane 2), 48 h (lane 3) and 312 h (lane 4), Lanes: M, isoelectric point marker; 1, purified last two maturation forms of the wild-type A. faecalis PA(pI5.3andpI5.5). (B) Purified T206GS213G and T206GS213GT219G mutants were incubatedin1m M Tris/HCl pH 7.5 at 25 °Cfor0h(lanes1and4) and 192 h (lanes 2 and 5). Purified last two maturation forms of the wild-type A. faecalis PA (pI 5.3 and pI 5.5) served as references (lane 3). Fig. 5. Reversed Eadie–Hofstee plots for the hydrolysis of benzylpeni- cillin catalyzed by A. faecalis PA (pI 5.3) and A. faecalis PA mutants. Phosphate buffer pH 7.5, I ¼ 0.2 M ,25°C; substrate concentrations in the range 5 · 10 -6 to 80 · 10 )6 M ; enzyme concentrations in the range 3.2 · 10 )11 to 10 · 10 )11 M . The initial rates used to determine the steady-state kinetic parameters were average values of three inde- pendent experiments at each concentration. The standard deviations are given by error bars. 4726 V. Kasche et al. (Eur. J. Biochem. 270) Ó FEBS 2003 pro-sequence of A. faecalis PA revealed that residues Val216 to Lys226 are likely to adopt an a-helical confor- mation. Assuming a similar processing pathway as for E. coli PA (based on sequence homology, Fig. 1), the third residue for mutation, Thr219, was chosen to be a residue within the a-helix proportionally at the same position of the Asn241 in the a-helix of pro-peptide of E. coli PA. The processing phenotypes of all altered A. faecalis PA pro-peptide mutant precursors were analyzed by SDS/ PAGE (Fig. 3B). Introduction of an additional mutation at position 213 (T206GS213G) stabilized the precursor and in the processing patterns only PA-forms with longer A-chain (A + P*) corresponding to the 13 amino acids extension were detected (Fig. 3B, lane 3). Thus, the purified mutant appeared as a single stable band on the IEF-gels with a pI of 5.6 and was not further converted even after incubation at room temperature for 192 h (Fig. 4B). A third mutation in the pro-peptide at position 219 (T206GS213GT219G) showed quite diverse effects. The SDS-processing pattern of this mutant revealed an appearance of an unstable intermediate with a larger A-chain (A + P* form, Fig. 3B, lane2), which after 72 h is further converted to the A + P form (data not shown). This suggests that the introduced mutation at position 219 causes only retardation, and not complete blockage of this cleavage. Meanwhile, mutagenized Thr219 also seems to destabilize the peptide chain at the other exchanged (T206G and S213G) positions and a band corresponding to the completely processed A. faecalis PA (pI 5.3) was detected on the IEF gels, even immediately after purifi- cation (Fig. 4B, lanes 4, 5). Nevertheless, both pro-peptide mutants (T206GS213G and T206GS213GT219G) exhi- bited increased specific activities (1.9- and 2.3-fold, respectively) compared with the completely processed A. faecalis PA with pI 5.3 (Table 3). These results are in good agreement with the observed in vitro activation of A. faecalis PA (pI 5.3) by fragments of the pro-peptide with a corresponding length (11-mer and 20-mer) (Fig. 2). The k cat value for benzylpenicillin hydrolysis catalyzed by the T206GS213G mutant was higher than the value for the T206G mutant (Fig. 5). The introduced third mutation in the pro-peptide of A. faecalis PA in the T206GS213GT219G mutant resulted in a 2.9-fold increase of the specificity constant compared with A. faecalis PA, mainly due to the higher turnover number (Fig. 5). Pro-domains of many zymogenes have been shown to accelerate 3D-structure formation [31] or to influence the folding as an intramolecular chaperone [32,33]. The mech- anism by which fragments of a pro-peptide function as activating factors is presently unknown. Based on the results presented in this study, we assume that fragments of the pro-peptide of A. faecalis PA activate the enzyme by stabilizing the transition state of acyl-enzyme formation resulting in enhanced catalytic constants for all of the mutants with extended A-chains. Even though the observed activation of A. faecalis PA in cell homogenate has been explained by the results so far obtained, many questions remain to be answered: What is the biological significance in generating enzymes for which activity decreases in the maturation process? What is the molecular mechanism by which fragments of the pro-peptide exactly influence the catalytic constant of the enzyme? Acknowledgements We thank Dr Frank Meyberg, Institut fu ¨ r Anorganische und Angewandte Chemie, Universita ¨ t Hamburg, for performing the ICP- AES analyses. References 1. 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