Báo cáo Y học: Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato pptx

Identification 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 Zn
2+
-binding (Asp347,
Glu429) and two catalytic (Arg431, Lys354) residues in the
tomato leucine aminopeptidase (LAP-A) function was
tested. T he i mpact of substitutions at these positions,
corresponding to the bovine LAP residues A sp255, Glu334,
Arg336, and Lys262, was e valuated in His
6
–LAP-A fusion
proteins expressed in Escherichia coli. S ixty-five percent of
the mutant H is
6
–LAP-A proteins were unstable or h ad
complete or partial defects in hexamer assembly or stability.
The activity of hexameric His
6
–LAP-As on Xaa-Leu and
Leu-Xaa dipeptides was tested. Most substitutions of
Lys354 (a catalytic residue) resulted in His
6
–LAP-As that
cleaved d ipeptides at slower rates. The Glu429 mutants ( a
Zn
2+
-binding residue) had more diverse phenotypes. Some
mutations abolished activity and others retained partial o r

complete activity. The E429D His
6
–LAP-A enz yme had K
m
and k
cat
values similar to the wild-type His
6
–LAP-A. One
catalytic (Arg431) and one Zn-binding (Asp347) residue
were essential f or His
6
–LAP-A activity, as most R431 and
D347 mutant His
6
–LAP-As did not hydrolyze dipeptides.
The R431K His
6
–LAP-A that retained the positive c harge
had pa rtial ac tivity as reflected in the 4.8 -fold d ecrease in k
cat
.
Surprisingly, while the D347E mutant (that retained a neg-
ative charge a t position 347) was inactive, the D347R mutant
that introduced a positive charge retained partial activity. A
model to explain these d ata is proposed.
Keywords: aminopeptidases; wounding; d efense response;
proteolysis; exopeptidases.
Aminopeptidases catalyze the hydrolysis of N-terminal
amino-acid residues from peptides and proteins. B ased 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 w ith 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 feeding, 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
coli LAP (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]. P eptide substrates with N-terminal ( P1) Asp o r Gly
residues were cleaved inefficiently. When substrates with
N-terminal aromatic residues w ere evaluated, significant
differences in the plant, E. coli , a nd porcine LAPs were
noted [17]. Phe and Tyr in the P1 position of dipeptides
promoted dipeptide cleavage by the porcine LAP and
E. coli PepA, but reduced tomato LAP-A hydrolysis r ates
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 P lant
Sciences, University of California, Riverside, CA 92521–0124, USA.
Fax: + 909 787 4437, Tel.: + 909 787 4687,
E-mail: lw
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.
*Presen t address : Crop Improvement and Utilization Research Unit,
Western Regional Research Center, ARS, U SDA, Albany, CA 94710,
USA.
Note: web page available at http//cnas.ucr.edu/bps/walling.html
(Received 29 A ugust 2001, revised 16 January 2002, accepted 18
January 2002)
Eur. J. Biochem. 269, 1630–1640 (2002) Ó FEBS 2002
Lys and Gly slowed the hydrolysis rates of the tomato
LAP-A, porcine LAP, and E. coli PepA markedly. An alysis
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 no t surprising that there is substantial
identity in the primary sequence o f 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 or
L
-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 a nd 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 i n
catalysis.

Each LAP subunit h as two domains. T he larger lobe of
the LAP protomer binds two z inc ions in a nonequ ivalent
fashion [25]. The Zn ions are located at the edge of an eight-
stranded, saddle-shaped b shee t. 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 co nserved 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 L AP-A to hydrolyze
substrates with varying P 1 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 K

mand
k
cat
for two muta nt His
6
–LAP-As
(R431K and E335D) were compared with values for the
wild-type His
6
–LAP-A.
MATERIALS AND METHODS
Site-directed mutagenesis
The LapA1 cDNA clone (pBLapA-M) s pans 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 4 0 recombinant clones p er mutagenized
residue were screened for m utations using LapA1 gene-
specific primers and dideoxy-chain-termination DNA
sequencing. A random distribution of mutations was not
obtained. Therefo re, several specific oligonucleotide primers
were designed to generate additional mutations of interest
(Table 1).
Overexpression and purification of wild-type

His
6
–LAP-A and mutant His
6
–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. Th e bovine and E. coli LAP residues
implicated in catalysis and zinc ion binding are described by Stra
¨
ter et al. [22,24]. Corresp onding residues from the tomato LAP-A1 p rotein were
identified. Oligonucleotides used for L apA1 mutagenesis are shown, where N ¼ A , T, G, or C. The mutagenized codon i s underlined.
Predicted
role in LAP
LAP residue
Bovine E. coli Tomato Oligonucleotide sequence Mutants
Zn-binding D255 D275 D347 5¢ GGATTAACTTTT
NNNAGTGGTGGCTAC 3¢
5¢ GGATTAACTTTT
CGCAGTGGTGGCTAC 3¢
5¢ GGATTAACTTTT
AACAGTGGTGGCTAC 3¢
D347A, D347I, D347V, D347S,
D347G, D347Y, D347E
D347R
D347N
Catalysis K262 K282 K354 5¢ GGCTACAACCTC
NNNGTCGGAGCTCGT 3¢ K354M, K354G, K354T,
K354W, K354N, K354E,
K354R

Zn-binding E334 E354 E429 5¢ CAATACTGATGCT
NNNGGTAGGCTCACA 3¢
5¢ CAATACTGATGCT
GACGGTAGGCTCACA 3¢
5¢ CAATACTGATGCT
AGCGGTAGGCTCACA 3¢
E429A, E429V, E429G,
E429W, E429Q, E429R
E429D
E429S
Catalysis R336 R356 R431 5¢ TGATGCTGAGGGT
NNNCTCACACTTGC 3¢
5¢ TGATGCTGAGGGT
CAGCTCACACTTGC 3¢
R431A, R431V, R431G,
R431W, R431E, R431K
R431Q
Ó FEBS 2002 Site-directed mutagenesis of the tomato LAP-A (Eur. J. Biochem. 269) 1631
His residues. pQLapA-M expresses the mature LAP-A1
protein as a His
6
–LAP-A protein (wild-type His
6
–LAP-A)
in E. coli and was previously described [4]. LapA cDNA
inserts with mutations (above) were excised from t he
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 His

6
–LAP-A clones. The
methods to over-express and purify the wild-type and
mutan t His
6
–LAP-A proteins by Ni/nitrilotriacetic acid
resin columns (Qiagen) have been described [17]. A liquots
(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 ac tivity
using t he chromogenic substrate
L
-Leu-p-nitroanilide
(Leu-p-NA) [4]. His
6
–LAP-A multimeric complexes were
evaluated by native PAGE.
Enzyme activity assays
The activities of the wild-type and mutant His
6
–LAP-A
enzymes on peptide substrates were determined using the
assay d escribed 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 m
M

dipeptide in 42 m
M
sodium carbonate (pH 9 .2). In
a typical assay, 100 lL of substrate solution was mixed with
10 lI of wild-type o r mutant His
6
–LAP-A enzyme
(2.5 ngÆlL
)1
)and15lLof5m
M
MnCl
2
. The mixture
was incubated at 37 °C for 30 min, and the reaction was
stopped by adding 3 mL of fresh TNBS reagent (65 m
M
sodium tetraborate, 0.8 m
M
cupric sulfate, 0.96 m
M
TNBS).
After a 30-min incubation at room temperature, the
absorbance of TNB/amino-acid conjugates was measured
at A
420
.AstheA
420
of each TNB/amino-acid conjugate was
distinct, standard curves for each amino acid were deter-

mined using concentrations between 0.5 and 5.0 m
M
.A
correlation coefficient was calculated for each peptide to
determine t he moles of peptide hydrolyzed. Relative rates of
hydrolysis were corrected for the percentage of enzyme in
the active h exameric form, w hich was determined f rom
scanned gel images and image analysis using
ALPHAIMAGE
software (Alpha Innotech Corporation, San Leandro, CA,
USA).
All dipeptides were in t he
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 b y B arrett [31] was u tilized. 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 K
m
and k
cat
values of wildtype and mutant His
6

–
LAP-As were determined from the initial rat es of hydrolysis
of Leu-Gly at concentrations ranging from 0.5 to 8.0 m
M
at
optimum pH (9.2). A nonlinear least-squares r egression of
enzyme kinetics was used to deter mine K
m
and k
cat
[32]. The
k
cat
value was based on (a) the masses o f the wild-type
(55 6 90 Da), E429D (55 743 Da), and R431K (55 666 Da)
His
6
–LAP-As, (b) the fact that all enzyme p reparations
were greater than 95% pure, and (c) the percentage of His
6
–
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 P AGE 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) a nd destained in methanol/acetic acid/water (40 :
10 : 50, v/v/v). Immunoblots were p erformed 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 w ith
plant, prokaryotic and animal LAPs using the
PILEUP
program from the Wisconsin Genetics Group. The Solanum
tuberosum (potato) LAP (StLAP; X67845) [12], Arabidopsis
thaliana LAP ( 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; 1 LCPA) [22,38] were
included in this study.
The
RASMOL
program (Berkeley version) and the blLAP–
leucinal complex (1lan) was used to determine distances
between atoms in blLAP [22]. Distances from the C
a
atoms
of Asp255, Arg336, Lys262 and Glu334 to side-chain
residues, Zn ions or leucinal atoms were determined. In
addition, the average distance between the C
a
of Glu and its

carboxylate oxygen, C
a
of Asp a nd its carboxylate oxygen,
C
a
of Arg and its side-chain amines or guanidinium, and C
a
of Lys and its side-chain amine was determined by
measuring distances in the blLAP. These average distances
were dete rmined by measuring atomic distances in 35 Glu
residues, 23 A sp residues, 23 Arg r esidues, and 26 Lys
residues in the blLAP-leucinal X-ray crystal structure [22].
The distances between the C
a
of Asp255 and Asp255
carboxylate oxygens, Zn1, Zn2, and C-terminal oxygens of
leucinal were previously reported.
RESULTS
LAPs have a highly conserved carboxyl domains
The leucine aminopeptidases of eukaryotic and p rokaryotic
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)rangedfrom41to46%.Thealignmentofeight
1632 Y Q. Gu and L. L. Walling (Eur. J. Biochem. 269) Ó FEBS 2002
LAPs in this 148-residue region showed a strict conservation
of residues i mplicated in zinc-ion binding and catalysis

(Fig. 1) [1]. Futhermore, extended regions of sequence
identity were also seen. T he shading in Fig. 1 underesti-
mates the degree of similarity between these enzymes, as
only residues c onserved in a ll or 5/8 of the different LAPs
were highlighted. Sixty-five of the 148 residues were identical
in all e ight 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
Zn
2+
binding and catalysis were imbedded in the highly
conserved regions I, II and IV. Ten a dditional 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 His
6
–LAP-A proteins
The c ompelling sequence identities of the bovine and
prokaryotic LAPs suggested that the plant LAP may use
a c atalytic mechanism similar t o 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 Zn
2+
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 b y 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 His
6
–LAP-A have similar biochemical
properties [17]. Therefore, wild-type and 31 mutant LAP-A
proteins were expressed in E. coli as His
6
/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 His
6
–LAP-A proteins were produced
in most bacterial lysates (data not shown). F or most His
6
–

LAP-A clones, an abundant 55-kDa His
6
–LAP-A protein
was isolated by affinity chromatography (Fig. 2). Four
mutations of His
6
–LAP-A resulted in unstable His
6
–LAP-A
proteins. In the K354T mutant strain, both the 55-kDa
His
6
–LAP-A protein a nd a 40-kDa degradation product
were detected (Fig. 2B). The replacement of Arg431 with
glycine (R431G) influenced the stability of the His
6
–LAP-A
proteins, because only a stable 28-kDa His
6
–LAP-A
polypeptide was purified by nickel column chromatography
(Fig. 2D). Finally, the D347A and R431E substitutions
resulted in rapid ly degraded His
6
–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 His
6

–LAP-As
The plant, a nimal a nd 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 His
6
–LAP-As to assemble
into hexamers, purified wild-type and 29 mutant His
6
–LAP-
As were subjected to native-PAGE and gels were stained
with Coomassie Blue (Fig. 3).
Although the purified wild-type His
6
–LAP-A and the
mutant His
6
–LAP-As displayed in Fig. 3 had intact 55-kDa
protomers (Fig. 2), the ability of mutant H is
6
–LAP-As to
Fig. 1. Comparison of plant, prokaryotic and a nimal LAP peptide
sequences spanning residues proposed to be involved in Zn
2+
binding and
catalysis. The deduced amino -acid se quen ces for t he tom ato L AP-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 liste d in
Materials and methods. Gaps in aligned peptide sequences (dots) were
introduced to maximize similarities. Residues identical in all six LAP s
are highlighted in black; residues conserved in five of the eight LAPs
areshadedingrey.ResiduespredictedtohavearoleinZnionbinding
(m)andcatalysis(d) are in dicated [1,24]. Residues po stulated to in-
teract bestatin or amastatin are located in a hyd rophobic pocket or
cleft and are indicated in open circles ( s)[39].
Ó FEBS 2002 Site-directed mutagenesis of the tomato LAP-A (Eur. J. Biochem. 269) 1633
form stable hexamers varied markedly (Fig. 3). Similar to
previous studies, the wild-type His
6
–LAP-A assembled into
a 357-kDa hexamer in E. coli and His
6
–LAP-A monomers
were not detected after nickel column chromatography
(Figs 2 and 3). Over 65% o f the His
6
–LAP-A mutants
exhibited some impairment in hexamer assembly o r stabil-
ity. Eleven of the His
6
–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 His
6

–
LAP-As (D347Y and D347E) and five of the Lys354
mutan t His
6
–LAP-As (K354N, K354T, K354W, K354G,
and K354M) exhibited more complex staining patterns.
These mutant Hi s
6
–LAP-As were able t o assemble into
hexamers, however, faster migrating complexes contribu-
ting to different percentages of the purified His
6
–LAP-A
protein were also visualized. These additional bands may
represent dissociation products or assembly intermediates of
the hexameric His
6
–LAP-A. The remaining 10 mutant
His
6
–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 His
6
–LAP-A enzymes
on Xaa-Leu dipeptides
The mutan t His
6

–LAP-As that did not assemble into
hexamers (Fig. 3) had < 0.5% activity on the Leu-Leu
dipeptide compared t o the wild-type His
6
–LAP-A (data not
shown). These data supported previous observations that
only the hexameric tomato LAP-A is functional [4]. The
remaining 19 mutant H is
6
–LAP-As that a ssembled into
hexameric enzymes were useful for examining the impact of
substitutions of active site residues on His
6
–LAP-A activity
and specificity. The ability o f wild-type and mutant His
6
–
LAP-As to hydrolyze peptides with 10 different N-terminal
(P1) residues was evaluated by measuring the rate of
Xaa-Leu p eptide hydrolysis (Table 2). The wild-type
His
6
–LAP-A hydrolyzed Xaa-Leu substrates at different
rates ranging from 46 (Tyr-Leu) to 419 lmolÆmin
)1
Æ
mg protein
)1
(Leu-Leu) as previo usly observ ed [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 s imilarly charged residue
(glutamic acid; D347E) or a small nonpolar residue (glycine;
D347G) abolished His
6
–LAP-A activity on all 10 Xaa-Leu
dipeptides (Table 2). When Asp347 was r eplaced with the
Fig. 2. SDS/PAGE fractionation of wild-type and mutant His
6
–LAP-A
proteins over-expressed in E. coli. Total p roteins w ere isolated from
E. c oli strains after IPTG induction. Proteins were fractionated by
SDS/PAGE and stained with Coomassie Blue. E. coli strains expre s-
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)runinaparallellane.
Fig. 3. Native PAGE fractionation of wild-type and mutant His
6
–LAP-

A proteins over-ex pressed in E. coli. Total proteins were isolated from
E. c oli 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 t he residues D347 (Panel A), K354 (P anel 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).
1634 Y Q. Gu and L. L. Walling (Eur. J. Biochem. 269) Ó FEBS 2002
positively 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 His
6
–LAP-A activity was observed with o ther
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 His
6
–LAP-A
activity on all Xaa-Leu dipeptides tested (Table 2). In
contrast, t he 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 His
6
–LAP-A
activity. Finally, t he replace ment o f glutamic a cid with
aspartic acid (E429D) yielded a His
6
–LAP-A enz yme 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 ac tivity.
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] a nd E. coli PepA [24]. Amino-acid residu e coordi-
nates for the b ovine L AP and tomato LAP-A (in parentheses) a re
shown. Zn1 is coordinated by ca rbonyl oxygens of Glu334 ()429),
Asp255 ()347), and Asp332 ()427; very weakly), a backbone carbonyl
of Asp322, and a water molecule. Zn2 is coo rdinated by the carbonyl
oxygens of Glu334 ()429), Asp255 ()347), Asp273 ()366), the back-
bone amino g roup of L ys250 ( )342), and a water molecule. The amino
terminus of the P1 r esidue is teth ered by Z n2 and a c arboxyl oxygen of
Asp273 ()366), while the p eptide’s carboxylate oxygen interacts with
Zn1 and side-chain amine group of Lys262 ()354). The P1¢ backbone
amino group interacts w ith the b ackbone carbonyl of Leu360 ()455),
while a bicarbonate is mo de led i nto the active site, there is not known if
water, bic arbonate or a bihydroxide ion a cts as t he 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.

Table 2. Activity o f wild-type and m utant His
6
–LAP-A proteins on Xaa-Leu dipeptides. The r ate of Xaa-Leu dipeptide hydrolysis was determined and e xpressed as 100% of wild-type His
6
–LAP-A activity
(± standard d eviation). T he hydrolysis rates for the wild-typ e H is
6
–LAP-A are shown in lmolÆmin
)1
Æmg protein
)1
. Rates indicated as < 0.5% of the wild-type His
6
–LAP-A values were at the limit of detection.
Relative activities of D347E, K354M, and R431K d ata were corrected for percentage enzyme that was in a hexameric form (see Materials and methods); all other enzymes were 100% hexamer.
His
6
–LAP-A
enzyme
% Wild-type His
6
–LAP-A activity on dipeptides
Leu-Leu Ala-Leu Val-Leu Phe-Leu Trp-Leu Pro-Leu Met-Leu Thr-Leu Tyr-Leu His-Leu
Wild-type 100 100 100 100 100 100 100 100 100 100
419.3 ± 30.9 320.6 ± 20.5 281.6 ± 8.9 131.3 ± 15.2 214.3 ± 6.7 153.4 ± 6.2 345.3 ± 9.5 261.3 ± 22 45.7 ± 5.9 150.9 ± 9.0
D347G <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5
D347E <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5
D347R 3.0 ± 1.0 1.0 ± 0.5 1.9 ± 0.2 0.6 ± 0.4 <0.5 <0.5 1.8 ± 0.5 5.9 ± 1.0 <0.5 0.7 ± 0.1
K354M 3.1 ± 0.6 <0.5 0.9 ± 0.3 0.9 ± 1.0 1.0 ± 0.3 <0.5 1.9 ± 0.4 7.4 ± 0.3 1.2 ± 0.3 1.8 ± 0.6
K354R 4.0 ± 0.8 1.8 ± 0.3 0.9 ± 0.5 2.0 ± 0.7 2.0 ± 0.7 <0.5 2.8 ± 0.5 7.8 ± 1.2 1.8 ± 0.8 1.6 ± 0.2

E429W <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5
E429V 1.7 ± 0.7 2.6 ± 0.6 2.7 ± 0.3 1.1 ± 0.4 0.9 ± 0.7 1.5 ± 0.5 0.7 ± 0.3 0.9 ± 0.4 1.0 ± 0.2 2.0 ± 0.2
E429D 95.6 ± 1.9 87.1 ± 10.2 101.3 ± 6.9 84.6 ± 9.3 91.6 ± 9.6 86.2 ± 15 94.6 ± 10.2 105.7 ± 9.4 78.5 ± 5.7 81.4 ± 12
R431A <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5
R431K 5.6 ± 0.8 2.6 ± 0.6 8.3 ± 0.2 5.0 ± 0.7 7.7 ± 1.0 1.3 ± 0.4 4.9 ± 0.4 11.3 ± 1.4 7.4 ± 0.8 4.9 ± 0.2
Ó FEBS 2002 Site-directed mutagenesis of the tomato LAP-A (Eur. J. Biochem. 269) 1635
The 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 His
6
–LAP-A a ctivity 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 His
6
–LAP-A levels) (Table 2). The
replacement of Lys354 with the similarly charged Arg
residue (K354R) c reated an enzyme w ith activities only
slightly above the K354M mutant activities (Table 2).
The Arg431 d ata contrasted with t he Lys354 mutant
analyses. Replacement o f Arg431 with alanine (R431A)
(Table 2) and o ther 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 L eu-Leu (5.6% of w ild-type

His
6
–LAP-A activity). Similar hydrolysis rates were noted
with other Xaa-Leu dipeptide substrates (Table 2).
Activity of wild-type and mutant His
6
–LAP-A
enzymes on Leu-Xaa dipeptides
To determine if mutation o f active site residues a ltered the
hydrolysis of peptides with different P1¢ residues, the activity
of mutant His
6
–LAP-A enzymes on nine Leu-Xaa dipeptide
substrates were tested (Table 3). The rates o f w ild-type
His
6
–LAP-A hydrolysis of these peptides v aried 45-fold
(Table 3). The impact of mutations in the residues (E429
and D 347) implicated in coordinating Zn
2+
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. R etention 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 His
6
–LAP-A
was inactive. Unlike Arg431, the active site residue Lys354
accommodated several substitution s. All K 354 mutant
enzymes had residual activity on the Leu-Xaa dipeptides
(Table 3; data not shown). Relative to t he other L eu-Xaa
dipeptides, a ll K354 mutant His
6
–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 His
6
–LAP-A activity levels, respectively
(Table 3).
Kinetics of hydrolysis of wild-type and mutant
His
6
–LAP-A enzymes
The kinetic properties o f E429D, R431K and wild-type
His
6
–LAP-A enzymes using Leu-Gly as a substrate were
Table 3. Activity of wild-type and mutant His

6
–LAP-A prote ins o n L eu-Xa a dipe ptides . T he r ate o f L eu-X aa dip eptide h yd rolysi s w as de termined and data i s e xpress ed a t 1 00% o f w ild-type His
6
–LAP-A activity
(± SD). Hydrolysis rates for the wild-type His
6
–LAP-A are shown in lmolÆmin
)1
Æmg protein
)1
. Rates indicated as <0.5% of the wild-type His
6
–LAP-A were at the limit of detection. Relative activities of
D347E, K354M, and R431K data were corrected for percentage enzyme that was in a hexameric form (see Materials and methods); all other enzymes were 100% hexamer.
His
6
–LAP-A
enzyme
% Wild-type His
6
–LAP-A activity
Leu-Leu Leu-Phe Leu-Met Leu-Ser Leu-Gly Leu-Tyr Leu-Asn Leu-Asp Leu-Arg
Wild-type 100 100 100 100 100 100 100 100 100
419.3 ± 30.9 440.9 ± 29.5 325.4 ± 20.9 150.4 ± 19.3 160.0 ± 5.6 200.8 ± 10.2 141.6 ± 7.8 9.8 ± 2.1 112.3 ± 6.9
D347G <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5
D347E <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 0.9 ± 0.2 <0.5
D347R 3.0 ± 1.0 2.7 ± 0.5 5.3 ± 0.3 2.8 ± 1.2 0.8 ± 0.3 5.3 ± 0.6 1.4 ± 1.0 0.7 ± 0.3 <0.5
K354M 3.1 ± 0.6 2.7 ± 1.3 1.8 ± 0.4 <0.5 1.6 ± 0.3 10.7 ± 1.0 1.5 ± 0.3 1.2 ± 0.2 1.3 ± 0.9
K354R 4.0 ± 0.8 5.7 ± 0.2 2.3 ± 0.4 2.2 ± 0.4 1.2 ± 0.6 14.2 ± 1.5 3.7 ± 0.9 0.7 ± 0.1 2.9 ± 0.9
E429W <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 0.6 ± 0.2 <0.5

E429V 1.7 ± 0.7 1.1 ± 0.3 2.9 ± 0.1 1.3 ± 0.1 1.0 ± 1.0 4.5 ± 0.4 4.4 ± 0.6 0.8 ± 0.2 4.0 ± 0.2
E429D 95.6 ± 1.9 107.0 ± 4.5 87.7 ± 6.2 89.9 ± 2.4 83.6 ± 5.9 92.5 ± 7.0 84.6 ± 4.1 52.7 ± 7.1 92.4 ± 5.1
R431A <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5
R431K 5.6 ± 0.8 3.6 ± 0.4 6.0 ± 0.5 4.1 ± 0.2 12.5 ± 0.8 11.1 ± 1.7 11.0 ± 1.0 7.5 ± 1.0 6.2 ± 0.6
1636 Y Q. Gu and L. L. Walling (Eur. J. Biochem. 269) Ó FEBS 2002
determined. The E429D enzyme hydrolyzed both Xaa-Leu
and L eu-Xaa pept ides at 52–107% of wild-type H is
6
–LAP-A
rates (Tables 2,3). Consistent with these observations, the
E429D His
6
–LAP-A and wild-type His
6
–LAP-A had a
similar K
m
values of 2.0 and 1.8 m
M
, respectively. In
addition, the turnover constant (k
cat
) was similar for the
E429D His
6
–LAP-A (108 s
)1
) and wild-type His
6
–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 w ild-type activity on Leu-G ly. This change in
hydrolytic activity was correlated with a 2.9-fold change in
the K
m
of the R431K His
6
–LAP-A (5.1 m
M
)relativeto
wild-type His
6
–LAP-A. In addition, the R431K enzyme had
a fourfold reduction in the turnover k
cat
to 30 s
)1
.
DISCUSSION
Intensive biochemical and structural studies of the bovine
lens LAP a nd 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 s equence
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 Zn
2+
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 u se a c atalytic
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 i on binding (Asp347 and Glu429) and catalysis
(Lys354 and Arg431) were subjected to site-directed mut-
agenesis. These r esidues correlated to the bovine lens LAP
(blLAP) residues Asp255, Glu334, L ys262, a nd Arg3 36
(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 asse mbly or stability. With the se H is
6
–LAP-A
preparations, no His
6

–LAP-A activity was detected. These
data furth er supported the observation th at 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 His
6
–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 His
6
–
LAP-A, as the quantity of the His
6
–LAP-A hexamer was
reduced in the strains expressing these His
6
–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 L AP-A enzyme did not reveal any correlation
with the n ature of t he residue in the mutant His
6
–LAP-A
enzyme.
X-ray structures of the unliganded blLAP and

E. coli PepA, and of the blLAP complexed with bestatin,
amastatin,
L
-leucine phosphonic acid, and
L
-leucinal have
been resolved [18–24,39]. These studies also identified the
residues important for LAP catalysis and coordination of
the two zinc io ns in the a ctive site. The i mpact of the
mutations on the tomato LAP enzyme must be viewed in
the context o f 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, [22,2 4]); 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 Asp25 5 (Asp347) and a car boxyl ox ygen of Glu334
(Glu429). T he interact ions of Zn1 and the carboxyl oxygen
of Asp332 (Asp427) are weak. The peptide is further

stabilized at the active site by the interaction of t he
N-terminal amine of t he P1 residue with a c arboxylate
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 P 1¢ 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. coli PepA and blLAP and the influence of bicarbonate
on PepA hydrolysis of a chromogenic substrate [24] suggests
that bicarbonate may act as a g eneral base. Bicarbonate (or
bihydroxide ion) may accept a proton from the zinc-
bridging water and shuttle t his p roton t o 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 t o 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 f rom 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
6
–LAP-A activity on dipeptide
Ó FEBS 2002 Site-directed mutagenesis of the tomato LAP-A (Eur. J. Biochem. 269) 1637
substrates. Only the R431K mutant that retained the
positive charge at t he active site had partial activity. These
observations were supported by kinetic measurements using
the Leu-Gly dipeptide. The K
m
of the R431K enzyme was
threefold higher and the k
cat
decreased fourfold rela tive 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 n itrogen and/or terminal amines. This less
optimal Lys side chain interaction with bicarbonate may
influence t he ability to e xtract a proton f rom t he
Zn-coordinated water, which would i nfluence k
cat
.
The tomato His
6
–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 b ecause the R431E His

6
–LAP-
A protein did not accumulate in E. coli. While the R356A
PepA had measurable activity on the chromogenic Leu-p-
NA substrate, the R431A mutation abolished His
6
–LAP-A
activity on all 19 dipeptides. As hydrolysis was not evident
when peptides were used at 5.6 m
M
, kinetic analysis of the
tomato R 431A His
6
–LAP-A was not possible. However
because the residual activity of the R431A was < 0.5%
and hydrolysis rates of Leu-Leu a nd Leu-Gly by the wild -
type tomato enzyme are known [17], the k
cat
for the R431A
His
6
–LAP-A could be no greater than 0.5 s
)1
.Thisis
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 o f the tomato
His

6
–LAP-A. Most substitutions for Lys354 created His
6
–
LAP-A e nzymes with partial activity o n dipeptides.
Significantly, the K354R mutant that retained a positive
charge at position 354 did not have an enhanced His
6
–
LAP-A activity relative to other K354 substitution mu-
tants. These data suggested that the longer side chain of
Arg d id not provide proper positioning of the guanidinium
nitrogen or the t erminal 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 K
m
and k
cat
consistent with the role of Lys282 in PepA (blLAP Lys262)
in binding of the carboxyl oxygen of the P1 residue and
stabilization of the ge m-diolate transition state. The Ala
substitution mutant (K354A) of the tomato His
6
–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 a n alteration in Zn
2+
binding was not provided
here, it was clear that substitution of these acidic residues
had a debilitating effect on LAP-A activity. Substitutions o f
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 th e 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 His
6
–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 c onsistent with the fact t hat the
Glu429 mutant (E429D) had similar K
m
and k
cat
values
when compared to the wild-type His
6
–LAP-A enzyme.
Similar mutants in the E. coli PepA or blLAP have yet to be
evaluated.

Asp347 (blLAP Asp255) was also implicated in Zn
2+
binding and stabilization o f the gem-diolate interme diate
(14,16). The tomato Asp347 was essential for LAP activity
because mutations abolished hydrolysis of all dipeptides.
The inactivity o f the D347E His
6
–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 bin ding o f t he zinc-bridging
water and/or ability of Z n2 and Zn1 to bind the N-terminal
amine and carbonyl oxygens of the P1 residue of the
substrate, respectively. T he replacement of Asp347 may
more sev erely alter functions associated wit h Zn1. This
hypothesis is b ased 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 w ere also consistent with the surprising
observation that rep lacement of Asp347 with Arg produced
aHis
6
–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 P 1
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 C
a
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 C
a
in 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 t omato L AP-A1 m utants should
allow m any newly emergent questions to be addressed.
The surprising residual activity in the D347R His
6
–
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
His
6

–LAP-A enzymes and other s ubstitutions that alter
the Zn1 and Zn2 binding residues influence Zn
2+
content of e ach LAP protomer.
ACKNOWLEDGEMENTS
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 h is sonic ator. This work was supported b y a National
Science Foundation Grant IBN-9318260 and IBN-0077862 (to
L. L. W.).
1638 Y Q. Gu and L. L. Walling (Eur. J. Biochem. 269) Ó FEBS 2002
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