One catalytic Arg431 and one Zn-binding Asp347 residue were essential for His6–LAP-A activity, as most R431 and D347 mutant His6–LAP-As did not hydrolyze dipeptides.. To determine if sub
Trang 1Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato
Yong-Qiang Gu* and Linda L Walling
Department of Botany and Plant Sciences, University of California, Riverside, CA, USA
The importance of two putative Zn2+-binding (Asp347,
Glu429) and two catalytic (Arg431, Lys354) residues in the
tomato leucine aminopeptidase (LAP-A) function was
tested The impact of substitutions at these positions,
corresponding to the bovine LAP residues Asp255, Glu334,
Arg336, and Lys262, was evaluated in His6–LAP-A fusion
proteins expressed in Escherichia coli Sixty-five percent of
the mutant His6–LAP-A proteins were unstable or had
complete or partial defects in hexamer assembly or stability
The activity of hexameric His6–LAP-As on Xaa-Leu and
Leu-Xaa dipeptides was tested Most substitutions of
Lys354 (a catalytic residue) resulted in His6–LAP-As that
cleaved dipeptides at slower rates The Glu429 mutants (a
Zn2+-binding residue) had more diverse phenotypes Some
mutations abolished activity and others retained partial or
complete activity The E429D His6–LAP-A enzyme had Km and kcatvalues similar to the wild-type His6–LAP-A One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6–LAP-A activity, as most R431 and D347 mutant His6–LAP-As did not hydrolyze dipeptides The R431K His6–LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat Surprisingly, while the D347E mutant (that retained a neg-ative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity A model to explain these data is proposed
Keywords: aminopeptidases; wounding; defense response; proteolysis; exopeptidases
Aminopeptidases catalyze the hydrolysis of N-terminal
amino-acid residues from peptides and proteins Based on
peptide sequences and enzymatic properties, hexameric
leucine aminopeptidases (LAP; EC 3.4.11.1) are present in
plants, animals, and prokaryotes [1–4] Most animals,
prokaryotes and non-Solanaceous plants express a single
class of LAP protomer [5–7] In contrast, there are two
classes of LAP enzymes (LAP-A and LAP-N) in tomato
with distinct biochemical characters and responses to
devel-opmental and environmental cues [4,7–11] LAP-N has
55-kDa protomers with a neutral pI LAP-N levels do not
change during plant growth and development or in response
to environmental stress (C J Tu & L L Walling,
unpublished results, [7,9]) While LAP-N is detected in all
plants examined, LAP-A is detected only in a subset of the
Solanaceae (N Ly & L L Walling, unpublished results,
[7,12]) LapA mRNAs, proteins and activities are detected
during floral and fruit development, but not in vegetative
organs of the healthy plants [8,11] LapA is expressed in leaves in response to several environmental stresses inclu-ding water deficit, salinity stress, caterpillar feeinclu-ding, and mechanical wounding [8,13,14] The changes in LapA RNA and protein levels after treatments with systemin, jasmonic acid, and abscisic acid implicate the wound octadecanoid pathway in LapA regulation [8,15,16]
The tomato LAP-A was biochemically characterized recently and compared to the porcine LAP and Escherichia coliLAP (XerB or PepA) [4,17] The LAPs from the three kingdoms are similar, as all three LAP enzymes are hexameric metallopeptidases that have high temperature and pH optima and are inhibited by the potent amino-peptidase inhibitors amastatin and bestatin [3,4] Analysis of amino acyl-p-nitroanilide and -b-naphthylamide chromo-genic substrates showed that all three enzymes efficiently hydrolyze substrates with N-terminal Leu, Arg, and Met residues [4] Studies with dipeptides and tripeptides demon-strated that, in general, the plant, animal and prokaryotic LAPs prefer to hydrolyze nonpolar aliphatic (Leu, Val, Ile, and Ala), basic (Arg) and sulfur-containing (Met) residues [4,17] Peptide substrates with N-terminal (P1) Asp or Gly residues were cleaved inefficiently When substrates with N-terminal aromatic residues were evaluated, significant differences in the plant, E coli, and porcine LAPs were noted [17] Phe and Tyr in the P1 position of dipeptides promoted dipeptide cleavage by the porcine LAP and
E coliPepA, but reduced tomato LAP-A hydrolysis rates
by 65 and 83%, respectively Reciprocally, the bulkier aromatic residue Trp impaired dipeptide cleavage by the porcine and prokaryotic enzymes more than the tomato LAP-A The penultimate residue (P1¢) strongly influenced LAP-A hydrolysis of dipeptides and the magnitude of its effect was dependent on the P1 residue [17] P1¢ Pro, Asp,
Correspondence to L L Walling, Department of Botany and Plant
Sciences, University of California, Riverside, CA 92521–0124, USA.
Fax: + 909 787 4437, Tel.: + 909 787 4687,
E-mail: lwalling@citrus.ucr.edu
Abbreviations: LAP, leucine aminopeptidase; blLAP, bovine lens
LAP; IPTG, isopropyl thio-b- D -galactoside; Leu-p-NA,
L -leucine-p-nitroanilide; TNBS, 2,4,6-trinitrobenzene sulfonic acid;
Xaa, variable amino-acid residue.
*Present address: Crop Improvement and Utilization Research Unit,
Western Regional Research Center, ARS, USDA, Albany, CA 94710,
USA.
Note: web page available at http//cnas.ucr.edu/bps/walling.html
(Received 29 August 2001, revised 16 January 2002, accepted 18
January 2002)
Trang 2Lys and Gly slowed the hydrolysis rates of the tomato
LAP-A, porcine LAP, and E coli PepA markedly Analysis
of tripeptides showed that more diversity was tolerated in
the P2¢ position [17]
Given the similar properties of the plant, animal and
bacterial LAPs, it is not surprising that there is substantial
identity in the primary sequence of these LAPs [3,13] The
bovine lens LAP (blLAP) is well-characterized
biochemi-cally [1,3] X-ray crystal structures of the native enzyme and
the blLAP complexed with aminopeptidase inhibitors
(amastatin or bestatin) and transition state analogues
(L-leucine phosphonic acid orL-leucinal) have been studied
[3,18–22] More recently, X-ray crystal structures of the
native E coli PepA were resolved [23,24] Based on the
bovine and E coli data, several mechanisms for LAP action
on substrates have been proposed and specific residues have
been implicated in substrate and metal binding and in
catalysis
Each LAP subunit has two domains The larger lobe of
the LAP protomer binds two zinc ions in a nonequivalent
fashion [25] The Zn ions are located at the edge of an
eight-stranded, saddle-shaped b sheet In the blLAP, the freely
exchanging zinc ion (Zn1; Zn488) is coordinated by the
carboxylate oxygens of Asp255, Asp332 and Glu334, and
the carbonyl oxygen of Asp332 The second zinc ion (Zn2;
Zn489) is more deeply imbedded in the LAP protomer and
exchanges divalent cations more slowly The carboxylate
oxygens of Asp255, Asp273, Glu334, and backbone amino
group of Lys250 [1] Analysis of X-ray crystal structures for
the bovine and prokaryotic LAPs and a limited mutational
analysis of the E coli PepA have suggested Lys262 and
Arg336 have a role in catalysis [22,23]
The residues implicated in zinc binding and catalysis for
blLAP are conserved in the tomato LAP-A For this reason,
a site-directed mutagenesis strategy was pursued to
charac-terize the active site in the tomato LAP-A Four residues
(Asp347, Glu429, Arg431, and Lys354) in the tomato
LAP-A that corresponded to blLAP residues implicated in
metal coordination (Asp255, Glu334) and catalysis
(Arg336, Lys262) were targeted for study Seven to nine substitutions were made at each position The impact of each residue on LAP-A protein stability and LAP-A assembly into a hexamer were assessed To determine if substitutions altered the ability of LAP-A to hydrolyze substrates with varying P1 and P1¢ residues, the hydrolysis
of 10 Xaa-Leu and 9 Leu-Xaa dipeptide substrates by wild-type and mutant LAP-A enzymes was determined Meas-urements of Km and kcat for two mutant His6–LAP-As (R431K and E335D) were compared with values for the wild-type His6–LAP-A
M A T E R I A L S A N D M E T H O D S Site-directed mutagenesis
The LapA1 cDNA clone (pBLapA-M) spans the entire LapA1 coding region for the mature LAP-A1 peptide (corresponding to LAP-A residues 54–571) [4] pBLapA-M was digested with BamHI and HindIII and the 1.6-kb LapA fragment was cloned into BamHI/HindIII-digested pUC119 (pULAP-M) Site-directed mutagenesis was performed according to Kunkel et al [26] using the Muta-Gene Kit (Bio-Rad, Hercules, CA, USA) Most mutants were created
by using LapA oligonucleotide primers, which replaced the codon of interest with random nucleotides (NNN) (Table 1) Over 40 recombinant clones per mutagenized residue were screened for mutations using LapA1 gene-specific primers and dideoxy-chain-termination DNA sequencing A random distribution of mutations was not obtained Therefore, several specific oligonucleotide primers were designed to generate additional mutations of interest (Table 1)
Overexpression and purification of wild-type His6–LAP-A and mutant His6–LAP-As
The wild-type and mutant LAP-A1 proteins were over-expressed in E coli as fusion proteins with six N-terminal
Table 1 Oligonucleotides used to create substitutions at tomato LAP-A residues D347, K354, E429, and R431 The bovine and E coli LAP residues implicated in catalysis and zinc ion binding are described by Stra¨ter et al [22,24] Corresponding residues from the tomato LAP-A1 protein were identified Oligonucleotides used for LapA1 mutagenesis are shown, where N ¼ A, T, G, or C The mutagenized codon is underlined.
Predicted
role in LAP
LAP residue
Zn-binding D255 D275 D347 5¢ GGATTAACTTTTNNNAGTGGTGGCTAC 3¢
5¢ GGATTAACTTTTCGCAGTGGTGGCTAC 3¢
5¢ GGATTAACTTTTAACAGTGGTGGCTAC 3¢
D347A, D347I, D347V, D347S, D347G, D347Y, D347E D347R
D347N Catalysis K262 K282 K354 5¢ GGCTACAACCTCNNNGTCGGAGCTCGT 3¢ K354M, K354G, K354T,
K354W, K354N, K354E, K354R
Zn-binding E334 E354 E429 5¢ CAATACTGATGCTNNNGGTAGGCTCACA 3¢
5¢ CAATACTGATGCTGACGGTAGGCTCACA 3¢
5¢ CAATACTGATGCTAGCGGTAGGCTCACA 3¢
E429A, E429V, E429G, E429W, E429Q, E429R E429D
E429S Catalysis R336 R356 R431 5¢ TGATGCTGAGGGTNNNCTCACACTTGC 3¢
5¢ TGATGCTGAGGGTCAGCTCACACTTGC 3¢
R431A, R431V, R431G, R431W, R431E, R431K R431Q
Trang 3His residues pQLapA-M expresses the mature LAP-A1
protein as a His6–LAP-A protein (wild-type His6–LAP-A)
in E coli and was previously described [4] LapA cDNA
inserts with mutations (above) were excised from the
pULapA-M clones by digestion with BamHI and HindIII,
cloned into BamHI/HindIII-digested pQE11 (Qiagen,
Chatsworth, CA) and transformed into E coli JM109 to
generate the pQ series of mutant His6–LAP-A clones The
methods to over-express and purify the wild-type and
mutant His6–LAP-A proteins by Ni/nitrilotriacetic acid
resin columns (Qiagen) have been described [17] Aliquots
(3 lL) of each 1-mL Ni-nitrilotriacetic acid column fraction
were separated by SDS/PAGE and stained with Coomassie
Brilliant Blue Aliquots were also assayed for LAP activity
using the chromogenic substrate L-Leu-p-nitroanilide
(Leu-p-NA) [4] His6–LAP-A multimeric complexes were
evaluated by native PAGE
Enzyme activity assays
The activities of the wild-type and mutant His6–LAP-A
enzymes on peptide substrates were determined using the
assay described by Mikkonen [27] with a few modifications
described by Gu & Walling [4] The amino acids liberated by
the LAP enzymes were quantitated with
2,4,6-trinitroben-zene sulfonic acid (TNBS; Sigma, St Louis, MO, USA)
reagent containing cupric ions to block the reaction with the
peptide amino groups [28] The substrate solution contained
6.25 mMdipeptide in 42 mMsodium carbonate (pH 9.2) In
a typical assay, 100 lL of substrate solution was mixed with
10 lI of wild-type or mutant His6–LAP-A enzyme
(2.5 ngÆlL)1) and 15 lL of 5 mM MnCl2 The mixture
was incubated at 37°C for 30 min, and the reaction was
stopped by adding 3 mL of fresh TNBS reagent (65 mM
sodium tetraborate, 0.8 mMcupric sulfate, 0.96 mMTNBS)
After a 30-min incubation at room temperature, the
absorbance of TNB/amino-acid conjugates was measured
at A420 As the A420of each TNB/amino-acid conjugate was
distinct, standard curves for each amino acid were
deter-mined using concentrations between 0.5 and 5.0 mM A
correlation coefficient was calculated for each peptide to
determine the moles of peptide hydrolyzed Relative rates of
hydrolysis were corrected for the percentage of enzyme in
the active hexameric form, which was determined from
scanned gel images and image analysis usingALPHAIMAGE
software (Alpha Innotech Corporation, San Leandro, CA,
USA)
All dipeptides were in the L-configuration Dipeptides
were ordered from Sigma Chemical Co or Bachem
(Torrance, CA, USA) Protein concentrations were
deter-mined by the Bradford assay [29] with bovine serum
albumin as a standard To facilitate discussion of the
specificity of the LAP enzymes, the nomenclature initially
developed by Schechter and Berger [30] and more recently
described by Barrett [31] was utilized Aminopeptidases
cleave between the N-terminal residue (P1) and the
penul-timate residue (P1¢) The C-terminal residue in tripeptides is
in the P2¢ position
The Km and kcat values of wildtype and mutant His6–
LAP-As were determined from the initial rates of hydrolysis
of Leu-Gly at concentrations ranging from 0.5 to 8.0 mMat
optimum pH (9.2) A nonlinear least-squares regression of
enzyme kinetics was used to determine K and k [32] The
kcat value was based on (a) the masses of the wild-type (55 690 Da), E429D (55 743 Da), and R431K (55 666 Da) His6–LAP-As, (b) the fact that all enzyme preparations were greater than 95% pure, and (c) the percentage of His6– LAP-A in hexameric form Enzyme purity and assembly (see above) was based on staining of SDS or native PAGE gels, respectively
SDS and native PAGE and immunoblot analyses SDS/PAGE was carried out according to the method of Laemmli [33] using a 10% polyacrylamide resolving gel and
a 4% stacking gel Native PAGE was performed according
to Gu et al [9] using a 7.5% polyacrylamide resolving gel and a 4% stacking gel Gels were stained with Coomassie Brilliant Blue in methanol/acetic acid/water (40 : 10 : 50, v/v/v) and destained in methanol/acetic acid/water (40 :
10 : 50, v/v/v) Immunoblots were performed as described
by Gu et al [9] The tomato LAP-A polyclonal antiserum does not significantly cross react with E coli proteins [4]
Sequence alignments The tomato LAP-A1 (LeLAP-A1; U50151) peptide sequence was previously reported [34] and was aligned with plant, prokaryotic and animal LAPs using the PILEUP program from the Wisconsin Genetics Group The Solanum tuberosum(potato) LAP (StLAP; X67845) [12], Arabidopsis thalianaLAP (AtLAP; P30184) [35], Petroselinum crispum (parsley) LAP (PcLAP; X99825), E coli PepA (EcPepA; P11648) [36], Rickettsia prowazekii PepA (RpPepA; P27888) [37], human LAP (HsLAP; AAD17527), and bovine mature, kidney LAP (BtLAP; 1LCPA) [22,38] were included in this study
TheRASMOLprogram (Berkeley version) and the blLAP– leucinal complex (1lan) was used to determine distances between atoms in blLAP [22] Distances from the Caatoms
of Asp255, Arg336, Lys262 and Glu334 to side-chain residues, Zn ions or leucinal atoms were determined In addition, the average distance between the Caof Glu and its carboxylate oxygen, Caof Asp and its carboxylate oxygen,
Caof Arg and its side-chain amines or guanidinium, and Ca
of Lys and its side-chain amine was determined by measuring distances in the blLAP These average distances were determined by measuring atomic distances in 35 Glu residues, 23 Asp residues, 23 Arg residues, and 26 Lys residues in the blLAP-leucinal X-ray crystal structure [22] The distances between the Ca of Asp255 and Asp255 carboxylate oxygens, Zn1, Zn2, and C-terminal oxygens of leucinal were previously reported
R E S U L T S LAPs have a highly conserved carboxyl domains The leucine aminopeptidases of eukaryotic and prokaryotic organisms shared a high degree of primary sequence identity
in the carboxyl region Alignment of C-terminal domain of eight LAPs is shown in Fig 1 The region spanning the tomato LAP-A residues 323–571 had between 76 and 92% identity with the Arabidopsis, potato, and parsley LAPs Identities with nonplant LAPs (bovine, human, E coli, and Rickettsia) ranged from 41 to 46% The alignment of eight
Trang 4LAPs in this 148-residue region showed a strict conservation
of residues implicated in zinc-ion binding and catalysis
(Fig 1) [1] Futhermore, extended regions of sequence
identity were also seen The shading in Fig 1
underesti-mates the degree of similarity between these enzymes, as
only residues conserved in all or 5/8 of the different LAPs
were highlighted Sixty-five of the 148 residues were identical
in all eight LAPs (43.9% identity) Most of these residues
were clustered into nine regions of identity (I–IX)
Inspec-tion of these regions showed that residues implicated in
Zn2+binding and catalysis were imbedded in the highly
conserved regions I, II and IV Ten additional residues
located in hydrophobic pockets and clefts have been
postulated to be important in van der Waal interactions
or hydrogen bonding with the aminopeptidase inhibitors,
bestatin and amastatin [39,40] These residues were located
in regions II, IV, V, VII, VIII and IX While many of these
residues were invariant, some were changes were noncon-servation substitutions changing the charge or polarity of residues (Fig 1) Finally, while regions III and VI were highly conserved, the role(s) of these conserved residues in LAP structure and/or function is not presently known
Over-expression and purification of wild-type and mutant His6–LAP-A proteins
The compelling sequence identities of the bovine and prokaryotic LAPs suggested that the plant LAP may use
a catalytic mechanism similar to that employed by the animal and bacterial LAPs [22,24] Using site-directed mutagenesis, a series of LAP-A1 mutants were generated (Table 1) The tomato residues with putative roles in Zn2+ binding (Asp347 and Glu429) and catalysis (Lys354 and Arg431) were substituted with residues that had the same or
a different charge, were isosteric, or had different degrees of hydrophobicity or hydrophilicity The tomato LAP-A1 residue designations are used throughout The analogous bovine and E coli LAP residues are identified by subtract-ing 92 or 112, respectively, from the tomato LAP-A1 residue number
Previous studies showed that the FPLC-purified tomato LAP-A1 and the His6–LAP-A have similar biochemical properties [17] Therefore, wild-type and 31 mutant LAP-A proteins were expressed in E coli as His6/fusion proteins After IPTG induction of bacterial cultures, total proteins were isolated, purified from soluble protein fractions using nickel column chromatography, and fractionated by SDS/ PAGE Coomassie Blue-stained gels and immunoblot analyses using a tomato LAP-A antiserum [9] revealed that high levels of soluble His6–LAP-A proteins were produced
in most bacterial lysates (data not shown) For most His6– LAP-A clones, an abundant 55-kDa His6–LAP-A protein was isolated by affinity chromatography (Fig 2) Four mutations of His6–LAP-A resulted in unstable His6–LAP-A proteins In the K354T mutant strain, both the 55-kDa His6–LAP-A protein and a 40-kDa degradation product were detected (Fig 2B) The replacement of Arg431 with glycine (R431G) influenced the stability of the His6–LAP-A proteins, because only a stable 28-kDa His6–LAP-A polypeptide was purified by nickel column chromatography (Fig 2D) Finally, the D347A and R431E substitutions resulted in rapidly degraded His6–LAP-A proteins that were not detected in E coli crude lysates or after affinity chromatography (data not shown)
Quaternary structure of wild-type LAP-A and mutant His6–LAP-As
The plant, animal and prokaryotic LAPs have multimeric structures [9,41,42] Similar to the bovine lens LAP, the tomato wound-induced LAP-A is a hexameric enzyme composed of six identical 55-kDa subunits [9] and the multimeric structure is critical for the enzyme activity [4] To investigate the ability of mutant His6–LAP-As to assemble into hexamers, purified wild-type and 29 mutant His6
–LAP-As were subjected to native-PAGE and gels were stained with Coomassie Blue (Fig 3)
Although the purified wild-type His6–LAP-A and the mutant His6–LAP-As displayed in Fig 3 had intact 55-kDa protomers (Fig 2), the ability of mutant His–LAP-As to
Fig 1 Comparison of plant, prokaryotic and animal LAP peptide
sequences spanning residues proposed to be involved in Zn 2+ binding and
catalysis The deduced amino-acid sequences for the tomato LAP-A1
(Le; residues 323–571), potato LAP (St; residues 304–554), Arabidopsis
LAP (At; residues 269–520), parsley LAP (Pc; residues 44–295); E coli
PepA (Ec; residues 251–503), R prowazekii (Rp; residues 247–500),
human LAP (Hs; residues 263–519), and mature bovine-kidney LAP
(Bt; residues 231–487) are shown Sequence accessions are listed in
Materials and methods Gaps in aligned peptide sequences (dots) were
introduced to maximize similarities Residues identical in all six LAPs
are highlighted in black; residues conserved in five of the eight LAPs
are shaded in grey Residues predicted to have a role in Zn ion binding
(m) and catalysis (d) are indicated [1,24] Residues postulated to
in-teract bestatin or amastatin are located in a hydrophobic pocket or
cleft and are indicated in open circles (s) [39].
Trang 5form stable hexamers varied markedly (Fig 3) Similar to
previous studies, the wild-type His6–LAP-A assembled into
a 357-kDa hexamer in E coli and His6–LAP-A monomers
were not detected after nickel column chromatography
(Figs 2 and 3) Over 65% of the His6–LAP-A mutants
exhibited some impairment in hexamer assembly or
stabil-ity Eleven of the His6–LAP-A substitution mutants
(E429W, E429V E429D, E429S, D437G, D347R, K354R,
R431V, R431Q, R431W and R431A) assembled into stable
hexameric complexes Two of the Asp347 mutant His6–
LAP-As (D347Y and D347E) and five of the Lys354
mutant His6–LAP-As (K354N, K354T, K354W, K354G,
and K354M) exhibited more complex staining patterns
These mutant His6–LAP-As were able to assemble into
hexamers, however, faster migrating complexes
contribu-ting to different percentages of the purified His6–LAP-A
protein were also visualized These additional bands may
represent dissociation products or assembly intermediates of
the hexameric His6–LAP-A The remaining 10 mutant
His6–LAP-As (E429G, E429Q, E429R, E429A, D347I,
D347S, D347V, D347N, K354E and R431G) did not form
stable hexamers, for only the fast-migrating dissociation
products were observed in native-PAGE gels (Fig 3)
Activity of wild-type and mutant His6–LAP-A enzymes
on Xaa-Leu dipeptides
The mutant His6–LAP-As that did not assemble into
hexamers (Fig 3) had < 0.5% activity on the Leu-Leu
dipeptide compared to the wild-type His6–LAP-A (data not
shown) These data supported previous observations that
only the hexameric tomato LAP-A is functional [4] The
remaining 19 mutant His6–LAP-As that assembled into hexameric enzymes were useful for examining the impact of substitutions of active site residues on His6–LAP-A activity and specificity The ability of wild-type and mutant His6– LAP-As to hydrolyze peptides with 10 different N-terminal (P1) residues was evaluated by measuring the rate of Xaa-Leu peptide hydrolysis (Table 2) The wild-type His6–LAP-A hydrolyzed Xaa-Leu substrates at different rates ranging from 46 (Tyr-Leu) to 419 lmolÆmin)1Æ
mg protein)1 (Leu-Leu) as previously observed [17] Although all 19 mutants were analyzed, data for two or three representative D347, K354, E429, and R431 mutants are presented (Table 2)
The aspartic acid residue at position 347 has a postulated role in coordinating both Zn1 and Zn2 (Table 1, [40]) Substitution of Asp347 with a similarly charged residue (glutamic acid; D347E) or a small nonpolar residue (glycine; D347G) abolished His6–LAP-A activity on all 10 Xaa-Leu dipeptides (Table 2) When Asp347 was replaced with the
Fig 2 SDS/PAGE fractionation of wild-type and mutant His 6 –LAP-A
proteins over-expressed in E coli Total proteins were isolated from
E coli strains after IPTG induction Proteins were fractionated by
SDS/PAGE and stained with Coomassie Blue E coli strains
expres-sing the wild-type His 6 –LAP-A (Panel C) or mutant His 6 –LAP-A
proteins with substitutions for the residues D347 (Panel A), K354
(Panel B), E429 (Panel C), and R431 (Panel D) are displayed The
His 6 –LAP-A protein sizes were determined by marker proteins (in
kDa) run in a parallel lane.
Fig 3 Native PAGE fractionation of wild-type and mutant His 6
–LAP-A proteins over-expressed in E coli Total proteins were isolated from
E coli strains after IPTG induction Proteins were fractionated by native-PAGE and stained with Coomassie Blue E coli strains expres-sing the wild-type or mutant His 6 –LAP-A proteins with substitutions for the residues D347 (Panel A), K354 (Panel B), E429 (Panel C), and R431 (Panel D) are displayed The mass of the wild-type His 6 –LAP-A was previously determined [17] and is shown at the left of each panel (357 kDa).
Trang 6positively charged arginine (D347R), the impact on LAP
activity was variable The D347R mutation abolished
hydrolysis of the Trp-Leu, Pro-Leu and Tyr-Leu substrates
Surprisingly, between 0.6% (Phe-Leu) and 5.9% (Thr-Leu)
of wild-type His6–LAP-A activity was observed with other
Xaa-Leu dipeptide substrates
Glu429 is also proposed to have a role in coordination of
both Zn1 and Zn2 in each LAP subunit (Table 1 and [18])
E429 mutants had three phenotypes Replacement of
Glu429 with tryptophan (E429W) abolished His6–LAP-A
activity on all Xaa-Leu dipeptides tested (Table 2) In
contrast, the E429V (Table 2) and E429S (data not shown)
mutants retained partial activity on all Xaa-Leu dipeptides
ranging from 0.7 to 2.7% of the wild-type His6–LAP-A
activity Finally, the replacement of glutamic acid with
aspartic acid (E429D) yielded a His6–LAP-A enzyme that
retained greater than 95% of its activity on the Leu-Leu
peptide Rates of hydrolysis of other Xaa-Leu dipeptides
were not impaired or were greater than 79% of the
wild-type activity
Fig 4 Model for the tomato LAP-A active site The model of the
tomato LAP-A active site is based on mechanisms proposed for the
bovine LAP [22] and E coli PepA [24] Amino-acid residue
coordi-nates for the bovine LAP and tomato LAP-A (in parentheses) are
shown Zn1 is coordinated by carbonyl oxygens of Glu334 ( )429),
Asp255 ( )347), and Asp332 ()427; very weakly), a backbone carbonyl
of Asp322, and a water molecule Zn2 is coordinated by the carbonyl
oxygens of Glu334 ( )429), Asp255 ()347), Asp273 ()366), the
back-bone amino group of Lys250 ( )342), and a water molecule The amino
terminus of the P1 residue is tethered by Zn2 and a carboxyl oxygen of
Asp273 ( )366), while the peptide’s carboxylate oxygen interacts with
Zn1 and side-chain amine group of Lys262 ( )354) The P1¢ backbone
amino group interacts with the backbone carbonyl of Leu360 ( )455),
while a bicarbonate is modeled into the active site, there is not known if
water, bicarbonate or a bihydroxide ion acts as the general base in
LAP-A The bicarbonate may interact with the water spanning Zn1
and Zn2, and residues from Gly335 ( )430), Arg336 ()431), and
Leu360 ( )445) Amino-acid residues of LAP and Zn ions are shaded
for contrast Bond lengths are not to scale.
is6
is6
1 Æmg
1
Trang 7The tomato LAP-A1 residues Lys354 and Arg431 may facilitate catalysis of substrates and/or stabilization of the gem-diolate reaction intermediate (Table 1, [22]) With the exception of the K354R mutant, most of the K354 substitution mutants had an impaired ability to form hexamers (Fig 3B) However, none of the mutations completely abolished His6–LAP-A activity on all Xaa-Leu substrates For example, the K354M mutation abolished activity on Pro-Leu and Ala-Leu, but other Xaa-Leu dipeptide substrates were hydrolyzed at detectable rates 0.9– 7.4% of the wild-type His6–LAP-A levels) (Table 2) The replacement of Lys354 with the similarly charged Arg residue (K354R) created an enzyme with activities only slightly above the K354M mutant activities (Table 2) The Arg431 data contrasted with the Lys354 mutant analyses Replacement of Arg431 with alanine (R431A) (Table 2) and other residues (Gly, Trp, Val, Gln) (data not shown) abolished LAP activity However, substitution of Arg431 with lysine (R431K) created an enzyme that retained partial activity on Leu-Leu (5.6% of wild-type His6–LAP-A activity) Similar hydrolysis rates were noted with other Xaa-Leu dipeptide substrates (Table 2)
Activity of wild-type and mutant His6–LAP-A enzymes on Leu-Xaa dipeptides
To determine if mutation of active site residues altered the hydrolysis of peptides with different P1¢ residues, the activity
of mutant His6–LAP-A enzymes on nine Leu-Xaa dipeptide substrates were tested (Table 3) The rates of wild-type His6–LAP-A hydrolysis of these peptides varied 45-fold (Table 3) The impact of mutations in the residues (E429 and D347) implicated in coordinating Zn2+on hydrolysis
of Leu-Xaa peptides was similar to that seen for the Xaa-Leu peptides The D347G, D347E, and E429W enzymes were inactive on all Leu-Xaa dipeptide substrates tested, while E429V exhibited a diminished but significant activity
on each substrate (Table 3) The D347R enzyme retained the ability to hydrolyze eight of the nine Leu-Xaa substrates
at significantly higher rates than other D347 mutants However, D347R did not hydrolyze the Leu-Arg signifi-cantly Retention of the negative charge at position 429 (E429D) created enzymes with near wild-type activity on Leu-Xaa peptides (Table 3) The hydrolysis of Leu-Asp was most strongly impaired in the E429D mutant
R431K enzyme that retained the positive charge at the catalytic site retained partial activity (3.6–13%) on Leu-Xaa peptides (Table 3) In contrast, the R431A His6–LAP-A was inactive Unlike Arg431, the active site residue Lys354 accommodated several substitutions All K354 mutant enzymes had residual activity on the Leu-Xaa dipeptides (Table 3; data not shown) Relative to the other Leu-Xaa dipeptides, all K354 mutant His6–LAP-A enzymes had an enhanced rate of cleavage of the Leu-Tyr peptide For example, the K354R and K354M enzymes retained 14 and 11% wild-type His6–LAP-A activity levels, respectively (Table 3)
Kinetics of hydrolysis of wild-type and mutant His6–LAP-A enzymes
The kinetic properties of E429D, R431K and wild-type His–LAP-A enzymes using Leu-Gly as a substrate were
is6
1 Æmg
1
is6
Trang 8determined The E429D enzyme hydrolyzed both Xaa-Leu
and Leu-Xaa peptides at 52–107% of wild-type His6–LAP-A
rates (Tables 2,3) Consistent with these observations, the
E429D His6–LAP-A and wild-type His6–LAP-A had a
similar Km values of 2.0 and 1.8 mM, respectively In
addition, the turnover constant (kcat) was similar for the
E429D His6–LAP-A (108 s)1) and wild-type His6–LAP-A
enzymes (120 s)1)
Unlike other Arg431 substitution mutants, the R431K
enzyme retained low levels of activity on all dipeptides with
13% of the wild-type activity on Leu-Gly This change in
hydrolytic activity was correlated with a 2.9-fold change in
the Km of the R431K His6–LAP-A (5.1 mM) relative to
wild-type His6–LAP-A In addition, the R431K enzyme had
a fourfold reduction in the turnover kcatto 30 s)1
D I S C U S S I O N
Intensive biochemical and structural studies of the bovine
lens LAP and E coli PepA provided a solid foundation for
the study into the residues critical of the activity of the
wound-induced tomato LAP-A1 [6,22,24] Similar to the
animal and prokaryotic enzymes, LAP-A1 is a
homo-hexameric enzyme [9,17] The alignment of 148 residues
from the C-terminal domain of seven different LAPs with
the tomato LAP-A1 showed a high degree of sequence
identity In addition to the strict conservation of residues
implicated in Zn ion binding and catalysis [1], more
extended regions of sequence identity were seen (regions
I–IX) The residues implicated in Zn2+binding, catalysis
and interactions with aminopeptidase inhibitors were
located in seven of these regions (I, II, IV, V, VII, VIII,
and IX) [39,40] The remaining conserved domains (regions
III and VI) make unknown contributions to structure
and/or function of the LAP enzymes
The compelling sequence identities in LAP enzymes
suggested that the tomato LAP-A may use a catalytic
mechanism similar to that employed by the animal and
bacterial LAPs [22,24] To test this hypothesis, four
residues in the tomato LAP-A1 protein with a potential
roles in Zn ion binding (Asp347 and Glu429) and catalysis
(Lys354 and Arg431) were subjected to site-directed
mut-agenesis These residues correlated to the bovine lens LAP
(blLAP) residues Asp255, Glu334, Lys262, and Arg336
(Table 1)
Thirty-one mutant enzymes were purified and analyzed
A large proportion (65%) of the mutant enzymes was
inherently unstable or had partial or complete defects in
hexamer assembly or stability With these His6–LAP-A
preparations, no His6–LAP-A activity was detected These
data further supported the observation that the hexameric
structure of LAP-A was essential for its enzymatic activity
[4,9] Several of the Asp347 mutants (D347Y and D347E)
and Lys354 mutants (K354N, K354T, K354W, K354G,
and K354M) assembled hexameric His6–LAP-A enzymes,
however, faster migrating complexes were also visualized
The faster migrating bands may represent a dissociation
product or an assembly intermediate of hexameric His6–
LAP-A, as the quantity of the His6–LAP-A hexamer was
reduced in the strains expressing these His6–LAP-A forms
These data stress that documentation of the ability of
mutant LAP protomers to assemble into a hexameric
structure is critical prior to evaluating impact of a mutation
on activity levels Inspection of the active site residue replacements that caused changes in the conformation of the tomato LAP-A enzyme did not reveal any correlation with the nature of the residue in the mutant His6–LAP-A enzyme
X-ray structures of the unliganded blLAP and
E coliPepA, and of the blLAP complexed with bestatin, amastatin, L-leucine phosphonic acid, andL-leucinal have been resolved [18–24,39] These studies also identified the residues important for LAP catalysis and coordination of the two zinc ions in the active site The impact of the mutations on the tomato LAP enzyme must be viewed in the context of these data The most recent mechanism proposed for blLAP and E coli PepA action is briefly reviewed using the residue designations for the blLAP (Table 1, [22,24]); the tomato LAP-A1 residues studied appear in parentheses A model of the reactive site of the tomato LAP based on the bovine LAP and E coli PepA appears in Fig 4
When a peptide substrate is bound by blLAP, both Zn1 (Zn488) and Zn2 (Zn489) are bound by six ligands to form imperfect, octahedral coordination geometries Zn2 is coordinated by carboxyl oxygens from Glu334 (Glu429), Asp273 (Asp366), and Asp255 (Asp347), the backbone amino group of Lys250 (Lys342), and the N-terminal amine
of the P1 residue of the peptide substrate A water molecule that bridges Zn1 and Zn2 is the sixth ligand In addition to the zinc-bridging water molecule and the carbonyl oxygen
of the P1 residue, Zn1 is bound by the two carboxyl oxygens
of Asp255 (Asp347) and a carboxyl oxygen of Glu334 (Glu429) The interactions of Zn1 and the carboxyl oxygen
of Asp332 (Asp427) are weak The peptide is further stabilized at the active site by the interaction of the N-terminal amine of the P1 residue with a carboxylate oxygen of Asp273 (Asp366), the carbonyl oxygen of the P1 residue with the side-chain amine of Lys262 (Lys354), and the backbone amino group of the P1¢ residue with the backbone carbonyl oxygen of Leu360 (Leu455)
There is no proteineous residue in the blLAP active site that can act as a nucleophile Therefore, the zinc-bridging water is proposed to have this function Early studies suggested a role for waters and the bihydroxide ion in the general base mechanism of blLAP [21,24] The recent identification of bicarbonate in the active site of the native
E coliPepA and blLAP and the influence of bicarbonate
on PepA hydrolysis of a chromogenic substrate [24] suggests that bicarbonate may act as a general base Bicarbonate (or bihydroxide ion) may accept a proton from the zinc-bridging water and shuttle this proton to the leaving group (the N-terminal P1¢ amine) [24] While the mechanism for generating the nucleophilic hydroxide from the zinc-bridging water differs in the models (with bicarbonate or three waters at the active site), the subsequent steps to form and resolve the gem-diolate intermediate are similar [22,24] The nucleophilic hydroxide ion attacks the carbon at the scissile bond to form a gem-diolate intermediate This intermediate is stabilized by continued interaction with Zn1, Zn2, residues from Lys262 (Lys354), Asp273 (Asp366), Leu360 (Leu455), and the bicarbonate ion (or bihydroxide ion)
If analogous to blLAP, the tomato Arg431 (blLAP Arg336) will bind the bicarbonate ion at the active site [24] Arg431 was essential for His–LAP-A activity on dipeptide
Trang 9substrates Only the R431K mutant that retained the
positive charge at the active site had partial activity These
observations were supported by kinetic measurements using
the Leu-Gly dipeptide The Kmof the R431K enzyme was
threefold higher and the kcatdecreased fourfold relative to
the wild-type enzyme The analogous mutation in the E coli
PepA (R356K) reduced the turnover number by 15-fold
[24] These data suggested that side-chain amine of Lys was
positioned to partially substitute for the functions of the Arg
guanidinium nitrogen and/or terminal amines This less
optimal Lys side chain interaction with bicarbonate may
influence the ability to extract a proton from the
Zn-coordinated water, which would influence kcat
The tomato His6–LAP-A Arg431 mutant data
com-pared favorably to studies with additional E coli PepA
Arg356 mutants [24] PepA was inactivated by the R356E
mutation but comparisons with the analogous tomato
mutant could not be made because the R431E His6
–LAP-A protein did not accumulate in E coli While the R356–LAP-A
PepA had measurable activity on the chromogenic
Leu-p-NA substrate, the R431A mutation abolished His6–LAP-A
activity on all 19 dipeptides As hydrolysis was not evident
when peptides were used at 5.6 mM, kinetic analysis of the
tomato R431A His6–LAP-A was not possible However
because the residual activity of the R431A was < 0.5%
and hydrolysis rates of Leu-Leu and Leu-Gly by the
wild-type tomato enzyme are known [17], the kcatfor the R431A
His6–LAP-A could be no greater than 0.5 s)1 This is
similar to the value measured for the E coli PepA R356A
mutation (0.227/ s)
The tomato LAP-A1 Lys354 (blLAP Lys262; PepA
Lys282) is the second residue implicated in catalysis This
residue was not essential for the activity of the tomato
His6–LAP-A Most substitutions for Lys354 created His6–
LAP-A enzymes with partial activity on dipeptides
Significantly, the K354R mutant that retained a positive
charge at position 354 did not have an enhanced His6–
LAP-A activity relative to other K354 substitution
mu-tants These data suggested that the longer side chain of
Arg did not provide proper positioning of the guanidinium
nitrogen or the terminal amines relative to the P1 carboxyl
oxygen of the peptide substrate Stra¨ter et al evaluated a
single mutation in analogous site of the E coli PepA
(K282A) [24] This mutation impacted both Km and kcat
consistent with the role of Lys282 in PepA (blLAP Lys262)
in binding of the carboxyl oxygen of the P1 residue and
stabilization of the gem-diolate transition state The Ala
substitution mutant (K354A) of the tomato His6–LAP-A
was not evaluated in the tomato LAP-A1 series of
mutations
The tomato residues Glu429 and Asp347 residues may
coordinate both Zn1 and Zn2 residues [14,16] While
evidence for an alteration in Zn2+binding was not provided
here, it was clear that substitution of these acidic residues
had a debilitating effect on LAP-A activity Substitutions of
the tomato Glu429 (blLAP Gly334) impaired, but did not
abolish, hydrolysis of Xaa-Leu and Leu-Xaa peptides The
substitutions at Glu429 eliminated one of the hydrogen
bonds that participated in the octahedral coordination
geometry for Zn2 and Zn1 Perhaps the other hydrogen
bonds were strong enough to retain the positioning of Zn1
and Zn2, the nucleophilic hydroxide, and substrate
Retention of charge at position 429 (E429D) produced a
highly active His6–LAP-A enzyme suggesting that despite the differences in side chain length, the Asp and Glu at position 429 were appropriately positioned to promote near wild-type function This is consistent with the fact that the Glu429 mutant (E429D) had similar Kmand kcatvalues when compared to the wild-type His6–LAP-A enzyme Similar mutants in the E coli PepA or blLAP have yet to be evaluated
Asp347 (blLAP Asp255) was also implicated in Zn2+ binding and stabilization of the gem-diolate intermediate (14,16) The tomato Asp347 was essential for LAP activity because mutations abolished hydrolysis of all dipeptides The inactivity of the D347E His6–LAP-A showed that the additional carbon in the Glu side chain may have positioned the Glu carboxyl oxygens too close to Zn1 and Zn2, thereby preventing the octahedral coordination geometry normally associated with Asp347 Zn1 and Zn2 interactions [22] This would indirectly influence the binding of the zinc-bridging water and/or ability of Zn2 and Zn1 to bind the N-terminal amine and carbonyl oxygens of the P1 residue of the substrate, respectively The replacement of Asp347 may more severely alter functions associated with Zn1 This hypothesis is based on the fact in the D431 mutants, the Zn2 would retain five of six productive coordination and Zn1 would be coordinated by only four strong and one potential, weak interaction
These data were also consistent with the surprising observation that replacement of Asp347 with Arg produced
a His6–LAP-A enzyme (D347R) with residual activity on many dipeptides Given the long side chain of Arg, it is unlikely that the Arg side-chain guanidinium or the terminal amines would be positioned correctly to coordinate with Zn1 or Zn2 However, it is possible that the Arg residue could be oriented to productively interact with the P1 carbonyl oxygen of the substrate and substitute for Zn1 function This is supported by bond distances deduced from the blLAP-leucinal crystal structure [22] In blLAP, the Ca
of Asp255 was approximately 6.64 and 6.54 A˚ from the carbonyl oxygens of leucinal (a transition state analogue) and the average distance from the Cain Arg to its side-chain amines was 6.36 A˚
Collectively, these data indicate that the tomato LAP-A1 utilizes a reaction mechanism similar to that employed by the bovine LAP and E coli PepA Contin-ued evaluation of the tomato LAP-A1 mutants should allow many newly emergent questions to be addressed The surprising residual activity in the D347R His6– LAP-A is of particular interest Future studies will evaluate the orientation of Arg in position 347 and determine if this basic residue is correctly positioned to bypass the role of Zn1 in substrate binding and catalysis
In addition, it will be important to address if the D347R His6–LAP-A enzymes and other substitutions that alter the Zn1 and Zn2 binding residues influence Zn2+ content of each LAP protomer
A C K N O W L E D G E M E N T S
We thank Dr M F Dunn, W S Chao, C J Tu, M Matsui, and other Walling laboratory members for helpful conversations and Dr B Hyman for use of his sonicator This work was supported by a National Science Foundation Grant IBN-9318260 and IBN-0077862 (to
L L W.).
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