Fragments of pro-peptide activate mature penicillin amidaseVolker Kasche, Boris Galunsky and Zoya Ignatova Institute of Biotechnology II, Technical University Hamburg-Harburg, Hamburg, G
Trang 1Fragments of pro-peptide activate mature penicillin amidase
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 (kcat/Km) for the hydrolysis
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 Ca2+ 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-chain raised the activ ity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly
by an increase of the turnover number (kcat)
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
colienzyme 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 pro-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)
Trang 2the 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 (kcat)
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 mM 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-typeA 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 28C in minimal M9 medium, containing 2.5 gÆL)1 glucose Six hours after induction with isopropyl thio-b-D-galactoside (0.5 mM) 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 mMTris 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 ServalytPrecotes 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, 25C, 125 lM 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 (DA380min)1ÆA280)1) Pure E coli
PA with a concentration 1 mgÆmL)1possesses an A280value
of 2.0 [20] The formula for recalculation of the acti-vity measured with the same substrate at 405 nm is:
DA405min)1¼ 0.94 · DA380min)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
Trang 3A faecalisPA or enzymes with point mutations in the
pro-peptide were incubated with different amounts of
phenyl-methanesulfonyl fluoride in phosphate buffer pH 7.5,
I¼ 0.2Mfor 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 25C and pH 7.5 (phosphate buffer I ¼ 0.2M)
The used substrate concentrations were 5, 10, 20, 40, 60 and
80 lM Enzyme concentration in the reaction mixture were
between 3.2· 10)11Mand 10· 10)11M 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 Km and kcat 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 purifiedA faecalis
penicillin amidase
The activation of A faecalis PA in vitro was tested with
chemically synthesized fragments of the pro-peptide
(11-mer, 20-(11-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 ) was incubated for
15 min at 25C in phosphate buffer pH 7.5 I ¼ 0.2Mwith the above oligopeptides in the concentration range 0–75 nM Then the mixture was subjected to activity measurements using NIPAB as a substrate
Results and discussion
Sequence alignment and comparison withE 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 identified in the E 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
Trang 4Possible 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-calissequence with an arginine which provides the necessary
side chain with a basic pKa 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 ofA 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, calcium ion coordinating residues The residues of the pro-peptides in both sequences are underlined.
Trang 5(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 (pI of 5.3) 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)1ÆA 280 )1
Wild-type (pI 5.3) 2.0 ± 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, 213and 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.
Trang 6Our 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 25C 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 Kmand kcatfor benzylpenicillin hydrolysis are summarized in Fig 5 Whereas the Kmvalues for benzyl-penicillin hydrolysis by A faecalis PA (pI 5.3) and T206G mutant were equal, the kcatvalue for T206G mutant was about 1.5-fold higher (Fig 5) The similarity in the Km 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 kcatvalues A similar effect was observed for cephalosporin acylase from Pseu-domonassp 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 (pI 5.3 and pI 5.5).
(B) Purified T206GS213G and T206GS213GT219G mutants were
incubated in 1 m M Tris/HCl pH 7.5 at 25 C for 0 h (lanes 1 and 4)
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)6M ; enzyme concentrations in the range 3.2 · 10)11to 10 · 10)11M 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.
Trang 7pro-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 coliPA (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 faecalisPA 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 kcat 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.
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