With oligosaccharides as substrates, a reduction in kcat, an increase in Kmand distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the w
Trang 1Human salivary a-amylase Trp58 situated at subsite )2 is critical for enzyme activity
Narayanan Ramasubbu1, Chandran Ragunath1, Prasunkumar J Mishra1, Leonard M Thomas2,
Gyo¨ngyi Gye´ma´nt3and Lili Kandra3
1
Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA;2Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, CA, USA;3Department of Biochemistry,
Faculty of Sciences, University of Debrecen, Hungary
The nonreducing end of the substrate-binding site of human
salivary a-amylase contains two residues Trp58 and Trp59,
which belong to b2–a2 loop of the catalytic (b/a)8 barrel
While Trp59 stacks onto the substrate, the exact role of
Trp58 is unknown To investigate its role in enzyme activity
the residue Trp58 was mutated to Ala, Leu or Tyr Kinetic
analysis of the wild-type and mutant enzymes was carried
out with starch and oligosaccharides as substrates All three
mutants exhibited a reduction in specific activity
(150–180-fold lower than the wild type) with starch as substrate With
oligosaccharides as substrates, a reduction in kcat, an increase
in Kmand distinct differences in the cleavage pattern were
observed for the mutants W58A and W58L compared with
the wild type Glucose was the smallest product generated
by these two mutants in the hydrolysis oligosaccharides;
in contrast, wild-type enzyme generated maltose as the
smallest product The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates The mutant W58L exhibited lower binding affinity at subsites)2, )3 and +2 and showed an increase in transglycosylation activity com-pared with the wild type The lowered affinity at subsites )2 and )3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary a-amylase
Keywords: salivary a-amylase; site-directed mutagenesis; subsite engineering; oligosaccharide hydrolysis; crystal structure
a-Amylases (a-1,4-D-glucan glucanohydrolases, EC 3.2.1.1)
are endoglucanases, widely distributed in all three domains
of life (Bacteria, Archaea and Eucarya), and catalyze
reactions such as hydrolysis and transglycosylation of
polysaccharides [1,2] These enzymes, belonging to the
glycoside hydrolase family 13 [3], possess very low overall
sequence similarity among the various members;
nonethe-less, in four small regions around the active site, the
members exhibit a strong sequence similarity [4–6] and
harbor the (b/a)8barrel topology [7] This small number of
conserved but critical short regions whose residues are lined
up along the surface of a deep cleft carries out substrate
binding and catalysis in a-amylases [2]
In humans, a-amylase is present in both salivary and
pancreatic secretions; the overall primary sequences of the
pancreatic and salivary a-amylases are highly homologous,
and exhibit a high level of structural similarity [8,9] Human salivary a-amylase (HSAmy) is monomeric, calcium binding protein with a single polypeptide chain of 496 amino acids [9] The structure of HSAmy consists of three domains: domain A (residues 1–99, 170–404), domain B (residues 100–169) and domain C (residues 405–496) The domain A adopts a (b/a)8 barrel structure bearing three catalytic residues Asp197, Glu233 and Asp300 The domain B occurs
as an excursion from domain A and contains one calcium-binding site Domain C forms an all b-structure and seems to be an independent domain with as yet unknown function [9]
The active site of HSAmy and mammalian a-amylases
is well established and is present in domain A as a deep V-shaped cleft [8–13] The active site of HSAmy is divided into glycone binding sites ()4, )3, )2, and )1) and aglycone binding sites (+1, +2 and +3) [13] These consecutive sites (whose nomenclature follows the currently accepted nomen-clature [14]), have been suggested to interact with substrate glucosyl residues with cleavage occurring between subsites )1 and +1 [9,15,16] Enzymatic subsite mapping has been used to characterize the number of recognized substrate residues and the individual subsite binding affinity for HSAmy [17] Using this method, the high and low affinity subsites, which control the productive binding modes in HSAmy has been determined Among these, the subsite)2
of the glycone binding site and +2 of the aglycone binding sites possessed the highest affinity [17]
Correspondence to Narayanan Ramasubbu, Department of Oral
Biology, C-634, MSB, UMDNJ, 185 South Orange Ave, Newark,
NJ 07103 USA Fax: + 1 973 9720705, Tel.: + 1 973 9720704,
E-mail: n.ramasubbu@umdnj.edu
Abbreviations: CNP, 2-chloro-4-nitrophenyl; G2, maltose;
G3, maltotriose; G4, maltotetraose; G5, maltopentaose; G6,
malto-hexaose; G7, maltoheptaose; HSAmy, human salivary a-amylase;
MPD, 2-methyl-2,4-pentanediol; PNP, p-nitrophenyl.
Enzyme: a-amylase (a-1,4- D -glucan glucanohydrolase) (EC 3.2.1.1).
(Received 9 March 2004, accepted 23 April 2004)
Trang 2Subsite mapping of the substrate-binding site has also
been reported based on the crystal structure of HSAmy in
complex with a pseudohexasaccharide inhibitor derived
from acarbose [13] This structure has provided the detailed
stacking and hydrogen bond interactions occurring at
subsites )4 through +2 The glucose moieties occupying
the subsites)1 through )4 are each involved in a number
of interactions with the protein atoms While subsite )1
interacts with domain A (Arg195, Asp197, Glu233, His299
and Asp300) and domain B residues (His101 and Leu165),
the subsite )4 interacts only with domain B residues
(Asn105, Asp147 and Ser163) These residues are dispersed
in the loops following the strands b2 through b7 In contrast,
subsites)2 and )3 interact with residues Trp58, Trp59 and
Gln63 (contained in a loop connecting b2 and a2) and
His305 (in mobile loop 304–310) Although the residues
Trp58 and Trp59 are present in a number of a-amylases of
the Eukarya family, there are a few enzymes with Ala at
position 58 and a Tyr at 59 ([18]; follow the links
Multi-alignments and then Eukaryota at http://www.quimica
urv.es/pujadas/AAMY/AAMY_01/)
The two aromatic residues, Trp58 and Trp59 interact
with the bound substrate to different extent [8–13] The
residue Trp59 is involved in a stacking with the 4-amino-4,
6-dideoxy glucose and the glucose moiety at subsites)3 and
)2, respectively, and a hydrogen bond interaction to the
glucose moiety at subsite)2 In contrast, Trp58 has no such
stacking interaction with sugar moieties either at subsite)3
or at)2 Interestingly, the residue Trp58 is juxtaposed in
such a way that it interacts with many protein atoms both
in unliganded and in complex structures of HSAmy and
other mammalian a-amylases [8–13] For instance,
hydro-phobic interactions with residues Trp59, His299 and His305
and a hydrogen bond interaction with Asp356 are dominant
around Trp58 (Fig 1) As Trp58 is located in the vicinity of
subsite)2 and this subsite had the highest binding affinity
among the glycone subsites [17], we investigated the role
of Trp58 in the activity of HSAmy For this,
mutants Trp58fi Ala (W58A), Trp58 fi Leu (W58L)
and Trp58fi Tyr (W58Y) were generated and their kinetic
properties were compared with wild type using starch and oligosaccharides (both labeled and unlabeled) as substrates The crystal structure analysis of uncomplexed W58L and acarbose-soaked W58A mutant enzymes were also determined to analyze the structural differences, if any that might be used to explain the kinetic behavior of the mutants
Materials and methods
General procedures All buffer reagents and other chemicals were obtained from Sigma Chemical Co The acarbose was a generous gift from Bayer The expression and purification of the recombinant proteins was carried out as previously described [19] All oligonucleotides used in this study were synthetic products purchased from Integrated DNA Technologies; the oligo-nucleotide sequences used in this study are given below Sequencing was performed at the DNA Sequencing Resource Center at the Rockefeller University, New York
Bacterial strain, media and plasmids Bac-To-Bac Baculovirus Expression System was used to generate recombinant and mutant proteins using procedures outlined previously [19,20] The following forward pri-mers (5¢-CCTTTCAGACCTXXXTGGGAAAGATAC-3¢, where XXX ¼ GCG, CTG, and TAC, respectively, for W58A, W58L and W58Y) and the corresponding reverse oligonucleotide primers used to create the mutants studied
in this paper For W59A and W59L, the forward primer was designed based on W58 mutation except for the position change (5¢-CCTTTCAGACCTTGGXXXGA AAGATAC-3¢, where XXX ¼ GCG, CTG, respectively, for W59A, and W59L) All primers were used in vector pFASTBAC1 (Invitrogen) into which HSAmy gene was cloned [19] The mutations were verified by nucleotide sequencing of the HSAmy cDNA using appropriate primer The plasmid pFASTBAC1 with mutant HSAmy was used
to transform into MAX EFFICIENCY DH10BACTM
Fig 1 Conformational space occupied by Trp58 in wild-type HSAmy The Trp58 site of the wild-type HSAmy crystallized with acarbose showing the interactions involving the Trp residue (PDB Code 1mfv) Note that the side chain of Trp58 enters into a hydrogen bond with the main chain of Asp356 Note that Asp300 is one of the three catalytic residues All other contacts are of hydrophobic nature The distances are given in Angstroms All structural figures were drawn using [48].
Trang 3(Invitrogen) cells that contained baculovirus genomic DNA
(bacmid) as well as a helper plasmid Transformed cells were
plated on Kanamycin, Gentamycin, Tetracycline, Bluo-gal
and IPTG-containing plates A single white colony was
cultured overnight and the high molecular recombinant
bacmid DNA was isolated and transfected into Sf9 cells
using CELLFECTIN ReagentTM(Invitrogen) After 72 h
of incubation at 28C in SF900II serum-free medium
(Invitrogen), recombinant baculovirus was harvested from
the medium Viral stocks were amplified by re-infection
into suspension culture of Sf9 cells at 28C with continuous
shaking at a speed of 140 r.p.m
Protein expression and purification
The proteins were isolated from Sf9 cell culture grown in
1 L of the medium by following protocol previously
established for native HSAmy [19] after observing 100%
cell death Briefly, cell debris was removed from a 5-day
postinfected medium, adjusted to pH 8.0 with NaOH After
centrifugation, the supernatant was further clarified by
passing through a 0.45 lMfilter (Corning Inc.) and the low
molecular weight proteins were removed by ultrafiltration
(Amicon Inc.) using a 30 kDa cut-off spiral cartridge The
medium was lyophilized and resuspended in 100 mMTris/
HCl, pH 8.0 Following dialysis against a buffer (5 mM
Tris/HCl, pH 8.0) containing 2 mMCaCl2, and
centrifuga-tion, the supernatant was applied to a 3· 13 cm DEAE-52
cellulose column (Whatman) Bound materials were eluted
from the column as previously described [19] Fractions
containing recombinant protein were pooled based on
SDS/PAGE [21] and Western blotting and dialyzed against
cold deionized water using Spectra/Por2 (MWCO of
12–14 000 Da; Spectrum Medical Industries, Inc.) and
lyophilized At this stage, the enriched enzymes were
subjected to a BioGel P60 size exclusion chromatography
following a procedure described previously [22] After
pooling the fractions containing the desired mutant enzymes
based on Western blotting, enzymes with greater than 99%
purity were obtained at approximately 5 mgÆL)1 of the
culture medium
The mass and purity of the enzymes were confirmed by
mass spectral analysis using Perspective Biosystems, a DE
Pro MALDI-TOF instrument equipped with a laser at
337 nm and operated with a positive or negative detection
with 6 kV acceleration potential Samples were analyzed in
delayed extraction linear mode, calibrated externally with
bovine serum albumin (Sigma Chemical Co.) All spectra
were the result of averaging 200 shots
Enzyme activity assays
Dinitrosalicylic acid assay was used for measuring the
starch-hydrolyzing activity of HSAmy and mutants at
25C for 3 min in 20 mM phosphate buffer (pH 6.9)
containing 6 mMNaCl using 1% soluble starch as substrate
[23] Kinetic measurements were carried out using
4-nitrophenyl-a-D-maltoheptaoside (G7-PNP; Boehringer
Mannheim) and p-nitrophenyl-a-D-maltopentaoside
(G5-PNP; Sigma) in a coupled assay with 20 UÆmL)1of yeast
a-glucosidase (Boehringer Mannheim) Kinetic parameters
were calculated using the initial velocities (v) obtained from
seven substrate concentrations [S] in the range of 0.078–
5 mM The concentration of the wild type was 2 nM and concentration of the mutants W58A, W58Y and W58L was
20 nM as determined from molar absorbance at 280 nm (26.1 for HSAmy) and/or BCA protein assay (Pierce) A typical reaction was carried out in 100 mMHEPES buffer (pH 7.1) containing 50 mM NaCl and 10 mM CaCl2 at
30C All experiments were carried out in triplicate and the average value is reported
Hydrolysis of maltooligosaccharides Assays measuring the products of oligosaccharide hydro-lysis were carried out using a Varian HPLC (ProStar) system equipped with a single port manual injector and a refractive index detector (model number 350) The product distribution of the hydrolysis of oligosaccharide substrates
by the wild-type and mutant enzymes was determined by HPLC analyses at a single substrate concentration (0.5 mM)
at room temperature In these experiments, the secondary attacks on products were avoided by analyzing the reaction
at time points wherein the conversion was < 20% The hydrolysates were analyzed using an analytical Dextropak column (100· 8 mm) to which a Novapak C18 Guard Pak precolumn module was attached (Waters) Water was used
as an eluent Integration of the HPLC profiles was carried out using Varian Star software (Version 5.51) The a-anomers of the oligosaccharides (maltotriose through maltoheptaose) were identified from the retention times of the products obtained by the hydrolysis of amylose In a typical run, a total reaction volume (200 lL) consisted of either an enzyme concentration of 60 nM (HSAmy) or
500 nM (W58A, W58Y and W58L), oligosaccharide at 0.5 mM (G3 through G7) in water The reaction mixture (20 lL) was injected in to the HPLC system after a specific interval (1–15 min) The product profile was analyzed based
on retention times of standards run under similar conditions without the addition of the enzyme The retention times of the oligosaccharides were also compared using a mixture of G3 through G7 at 0.5 mMeach separated using the same HPLC system The amount of each product formed was determined using the area under each peak and converting it
in to molar concentration using values obtained previously for the standards These measured data were used to calculate the action pattern of various HSAmy enzymes for
a given substrate
Hydrolysis of maltooligosaccharide glycosides Oligosaccharides labeled with 2-chloro-4-nitrophenyl moi-ety (CNP) were synthesized from b-cylcodextrin [24] Incubations of the various CNP-labeled oligosaccharides
in 25 mM glycerophosphate buffer (pH 7.0) containing
5 mMCa(OAc)2and 50 mMNaCl were carried out at 37C for 30, 40 and 60 min for W58L The reactions were initiated by the addition of enzyme (final concentration of 1.85 nM HSA and 18.8 nM for the mutant W58L) to the solution containing 1.0 mMof substrate Samples (20 lL) were taken at various time intervals and injected into the chromatographic column The products were separated on
a Spherisorb ODS2 5 lm column (250· 4.0 mm) with acetonitrile–water (13 : 87) as the mobile phase and at a
Trang 4flow rate of 1 mLÆmin)1at 40C using a Hewlett-Packard
1090 Series II liquid chromatograph equipped with a diode
array detector and an automatic sampler As noted above,
care was taken to exclude the secondary attack on the
products by obtaining the product ratios from the early
stages of hydrolysis wherein the conversion was always
< 10% The effluent was monitored for CNP-glycosides at
302 nm and the products of the hydrolysis were identified
by using relevant standards and analyzed using
ChemSta-tion software suite The measured hydrolysis data were used
to calculate the catalytic efficiencies of the enzymes
Structure determinations
Crystals of the mutants W58L, W58A were grown using
conditions previously described [9,25] All crystallization
experiments were conducted at room temperature A
protein concentration of 16 mgÆmL)1in 10 mMTris/HCl
(pH 9.0) containing 5 mM CaCl2was used The reservoir
solution contained 40% 2-methyl-2,4-pentanediol (MPD)
and the hanging drops consisted of 2 lL of protein and
2 lL of reservoir solution Diffraction quality crystals
appeared over a period of one to 4 weeks To obtain the
complexes with acarbose, these crystals were soaked with
acarbose (1 mMfinal concentration) in 40% MPD for 24 h
and used for data collection Diffraction data were collected
on a Mar Research imaging plate area detector system
(W58L) or on a Rigaku R-AXIS IV + image plate area
detector (W58A) using Cu Karadiation (1.5418 A˚)
gener-ated from a Rigaku RU200 rotating anode generator
operating at 50 kV and 100 mA The crystals were mounted
on loops (Hampton Research) and flash frozen to)170 C
in liquid nitrogen One hundred frames were measured with
a 1 oscillation to give 98–100% complete data to 2.0 A˚
(W58L) or 2.1 A˚ (W58A) The data frames were exposed
for 10 min each Intensity data were integrated, scaled and
reduced to structure factor amplitudes using HKL suite of
programs [26] Data collection statistics are given in Table 1
The unit cell parameters were found to be isomorphous with
those of the wild-type HSAmy [19]
The refinement of these solutions was carried out using
the CNS package [27] wherein cycles of rigid body
refinement, simulated annealing, positional and thermal B factor refinements were carried out Bulk solvent corrections were incorporated in the refinement protocols A test set consisting of 5% of reflections was used to monitor the Rfree behaviour Manual model rebuilding was carried out using TOM-FRODO [28] and O [29] The complete polypeptide chains of the mutants were examined with Fo-Fc, 2Fo-Fc and omit maps During this process, the mutant enzymes W58L and W58A clearly showed absence of side chain density for Trp The residues were changed to reflect the respective mutations and for the remainder of the refine-ment this enzyme was treated as such
At this stage, clear-cut continuous density corresponding
to the oligosaccharide ligand was observed in the active site region of only the W58A crystal soaked in acarbose at subsites )1, +1 and +2 However, no oligosaccharide atoms were included in the refinement until the refinement
of the protein reached convergence The identity of the sugar moieties (either 5-hydroxymethylchonduritol or 4-amino-4,6-dideoxy-a-D-glucose or glucose) was deduced from the presence or absence density for the hydroxyl group
of the side chain at position C5 in the ring [13] Additionally, difference density maps calculated by giving zero occupancy
to the O6 atoms were used to assist in the identifications The refinements were continued by the inclusion of the sugar atoms Further examination of the density maps revealed no additional binding sites in the complex The final rounds of refinement were carried out using maximum likelihood method as implemented in REF-MAC-5 of the CCP4 package [30] Solvent molecules were added using the arp/warp procedure [31] in the CCP4 package The validity of the water molecules were assessed
on the basis of the presence of a peak at least 3 r in the difference map, at least one hydrogen bond to a protein atom (N or O) or if the water molecules were part of a chain connecting protein atoms, and refinement of thermal factor less than 50 A˚2 Manual fitting was interspaced between refinements when necessary The programsPROCHECK[32], CCP4 and CNSwere used for model analysis of the final refined structures The coordinates and structure factors have been deposited with the Protein Data Bank [PDB codes are 1jxj (W58L) and 1nm9 (W58A complex)]
Table 1 Summary of diffraction data collection values and structure refinement statistics NA, not applicable.
Cell dimensions: a,b,c (A˚) 52.3 · 75.2 · 135.0 51.9 · 74.0 · 134.5 Resolution range (A˚) 65.9–2.0 42.6–2.1
Total/unique number of reflections 11 613/36 285 188 351/31 104
Completeness (%): overall/last shell a 98.3/95.0 99.7/99.7
Mean I/rI: overall/last shell 16.2/3.0 19.1/9.4
Number of protein/solvent/other atoms 3926/322/0 3926/293/212
Number of reflections used 34 452 29 481
B factor (A˚ 2 ): protein/solvent/other 17/23/NA 23/29/29
r.m.s deviations: bonds (A˚)/angles () 0.015/1.6 0.009/1.1
a Last shell: 2.07/2.0 A˚; 2.15–2.10 A˚ b Reflections in the test set (number/%): 1795/5.0; 1564/5.0.
Trang 5Kinetics studies of mutants
Replacements at position 58 were based on decreasing bulk
(Ala and Leu) or partial retention of aromatic character
(Tyr) All mutants gave as a single band in SDS/PAGE after
final purification and no isozyme corresponding to the
glycosylated a-amylase ( 62 kDa) was observed in either
SDS/PAGE or through mass spectral analysis [19] The
effect of the mutations on the hydrolysis of starch was
examined by comparing the specific activities for starch
hydrolysis (Table 2) For the mutants W58A, W58L or
W58Y, the specific activity is 150–180-fold lower compared
with the wild-type enzyme For smaller oligosaccharides
such as a p-nitrophenyl derivative of maltopentaoside
(G5-PNP) and maltoheptaoside (G7-(G5-PNP), the kcatvalues were
lower significantly Interestingly, although the Kmvalues for
the mutants were similar to the wild type for the substrate
G7-PNP, the corresponding Kmvalues for the substrate
G5-PNP were higher Thus, for the G5 substrate, there is an
increase (5-fold) in the Kmand a decrease in kcat
(30–500-fold) compared with HSAmy The kcat/Kmvalue for the two
mutants W58L and W58A, which have no aromatic ring, is
less than W58Y but similar to that obtained for the D300N
mutant of human pancreatic a-amylase [33] The kcat/Kmfor
the W58Y mutant, albeit lower than the wild type, is 10-fold
higher than either W58L or W58A suggesting that an
aromatic residue at this position might be necessary In
sharp contrast, the values for the position 59 mutants were
only approximately twofold lower compared with the wild
type for starch as well as G7-PNP as substrates Clearly, the
mutation of Trp58 affects the ground state binding of the
substrate and enzyme activity
Hydrolysis of maltooligosaccharides
Product distributions were determined by HPLC for the
wild type as well as all three mutants with several
oligosaccharides A typical chromatogram using G4 is
shown in Fig 2 The substrates were assayed at 0.5 mM,
which was used in the standard assay and in previous studies
[13,20] For each of the mutants, the sites of cleavage for a
given oligosaccharide and the ratio of the products formed were determined as described previously [20] The hydrolysis
of each oligosaccharide at a single concentration was monitored by means of HPLC with an aid of Dextropak column The Dextropak column is able to separate the two anomers of maltooligosaccharides containing three or more glucose units The retention times for the a- and b-anomers
of these oligosaccharides were deduced by first determining the retention time for the a-anomer using HPLC as described earlier [20] Briefly, amylose was used as substrate under similar conditions and the products were separated by HPLC The products of hydrolysis of maltooligosaccharides (G3-G7) were all composed of only a-anomers as HSAmy, like other a-amylases, is a retaining enzyme The retention times of the a-anomers of G3 through G7 thus obtained were used to identify the b-anomer and its retention time in the hydrolysis experiment using oligosaccharide substrates These values were used then in the analysis of the action pattern of the HSAmy enzymes This approach allowed us
to determine the site of cleavage in the various productive binding modes [12] The results obtained from this analysis are shown in Fig 3 in which the arrows indicate the site
of cleavage and the numbers reflect the percent cleavage attained at each point
The analyses were carried out at time points wherein the substrate consumption was less than 20% Maltotriose is a smaller substrate, which is very weakly cleaved by the wild-type enzyme and W58Y whereas both W58L and W58A cleaved it into glucose and maltose The production of glucose, which was observed in the hydrolysis of higher oligosaccharides as well, was a characteristic of the mutants W58A and W58L (Fig 2A) The amount of glucose produced was dependent upon the nature of the mutant Thus, W58A produced more glucose than W58L, which in turn produced more than W58Y In contrast, the wild type did not produce any detectable glucose for any of the substrates Comparison of the productive binding modes for the wild type with the mutants revealed that the number
of cleavage modes in the mutants is higher than the wild type for all substrates (Fig 3) The presence of the aromatic side chain in the mutant W58Y, results in binding modes closely resembling the wild type albeit with significantly lowered k /K values (Table 2) In contrast, the other two
Table 2 Parameters for the hydrolysis of starch and oligosaccharides All assays were performed at pH 7.1 Average kinetic errors in kinetic parameters: specific activity (± 2–5%) for HSAmy and 15–20% for the mutants; K m (± 7–10%) and k cat (± 5–7%) N.D., not determined.
Enzyme
Hydrolysis of
x-fold decrease in k cat /K m
for G5-PNP substrate compared with wild-type enzyme
Starch
specific activity
(UÆmg of protein)1)
Heptasaccharide (G7-PNP) Pentasaccharide (G5-PNP)
k cat
(s)1)
K m
(m M )
k cat /K m
(s)1Æm M )1 )
k cat
(s)1)
K m
(m M )
k cat /K m
(s)1Æm M ) HSAmy 66 212 175 0.27 648 131 0.35 372 1
W58A 350 0.43 0.39 1.1 0.60 1.70 0.353 1.09 · 10 3
W58L 356 0.43 0.20 2.1 0.26 1.55 0.168 2.31 · 10 3
W58Y 434 0.80 0.31 2.6 4.1 1.63 2.515 0.15 · 10 3
W59A 38 100 111 0.26 427 N.D N.D N.D.
W59L 34 415 75 0.16 468 N.D N.D N.D.
H305A a 5016 12 0.28 43 N.D N.D N.D –
a [37].
Trang 6mutants, W58L and W58A, lacking the aromatic residue
exhibit apparently altered binding modes
Hydrolysis of maltooliogosaccharide glycosides
The production of glucose in the hydrolysis by mutants
might have occurred in two different binding modes, where
the subsites from )4 to +1 were occupied or where the
subsites from)1 to +3 were occupied If the mutation of
the residue Trp58 affected binding at glycone subsites, use of
labeled substrates might provide additional insights into the
binding modes in these mutants For this purpose, we used
the mutant W58L and CNP-labeled substrates to determine
unambiguously the exact glycosidic linkage being cleaved
and the cleavage frequency A sample chromatogram is given in Fig 2B and the distribution of the products were calculated for a given substrate and summarized in Table 3 The product distribution for the W58L mutant is different from that of the wild-type HSAmy When CNP-G3 was used as the substrate productive binding modes in which)1 alone is occupied (leading to CNP-G2) or when )2 and )1 are occupied (leading to CNP-G1) occurs with equal frequency Three different binding modes are observed in the hydrolysis of G4 for both wild type and W58L but with an increase in the production of glucose from the nonreducing side only in W58L As seen with the unlabeled oligosaccharides (Fig 3), W58L generates: (a) more products from CNP-labeled oligosaccharides and (b)
Fig 2 HPLC analysis of the products of the reactions of the HSAmy and W5L enzymes with G4 oligosaccharide and CNP-G4 (A) HPLC analysis of the products of the reactions of the HSAmy enzymes with G4 oligosaccharide Note that oligosaccharides G3 or higher give rise to two peaks corresponding to the a-anomer (early eluting) or the b-anomer (late eluting) The products were identified using standards and amylose as described
in the Experimental section Note that glucose is generated by the mutant W58A as well as W58L (B) HPLC analysis of the products of the reaction
of the W58L enzyme with CNP-G4 As a result of increased transglycosylation activity of this enzyme significant amounts of CNP-G5, CNP-G6 and CNP-G7 are produced The wild-type enzyme did not show such activity Note the generation of CNP-G3, which suggests that in this cleavage mode, W58L generates glucose from the nonreducing end.
Trang 7more CNP-G or CNP-G2 than the wild type This suggests that higher population of the productive binding modes, in which subsite)1 alone or )2 and )1 at the nonreducing ends are occupied, occurs in the mutant W58L than in wild type Also, the binding at the subsites)3 and )4 might be affected by the mutation
The relative rate of formation of each product from the hydrolysis of a series of oligomeric substrates has been used
to estimate the subsite-binding energy in HSAmy and its mutants [17] Using this method the binding affinities for the four glycone and three aglycone binding sites in the mutant W58L, with the exception of the two subsites adjacent to the catalytic site, were calculated using a procedure suggested
by Allen and Thoma [34] The binding energies for the subsites)3, )2 and +2 are substantially lower compared with that of HSAmy (Fig 3B)
As Trp58 is situated at subsite)2 with the highest affinity among the glycone binding sites, its mutation affects the cleavage propensity of individual bonds in maltooligosac-charides (Fig 3A; Table 3) Reducing the bulk of the side chain at position 58 appears to suppress the binding beyond subsite )2 A reduction of the binding affinities in neigh-boring subsites is expected for multivalent ligands that bind
in a cooperative manner Thus, if one binding site shows reduced affinity, the neighboring binding sites will too, because the binding sites are not independent of each other Because of this reduction in the binding affinity at these subsites, there is an increase in the productive binding mode
in which glucose from the nonreducing end is generated; however, this is accompanied by a severe loss in activity (Table 2)
Structural studies of W58L and W58A Substitution of a Leu residue for Trp58 causes little perturbation in the structure (Fig 4A) The conformation
of the main chain and the orientation of the side chains of the active site residues are very close to those of the counterparts in the wild-type enzyme A notable exception is the region 304–310, the mobile surface loop, which adopts a completely different conformation (Fig 4B) This adapta-tion of a loop conformaadapta-tion is in accordance with the absence of substrate in the active site as has been shown previously in several wild-type a-amylases including HSAmy [8–10] However, W58L had clear electron density, except for His305, for the entire loop (Fig 4C) unlike wild-type a-amylase structures that exhibited only weak density
in this region [8–10] The substitution of the bulkier Trp with a shorter nonaromatic side chain leads to more open space in the vicinity of the Leu58 site However, this void in the mutant is unoccupied with any water molecules Another interesting feature about the W58L structure is the absence of a hydrogen bond between the catalytic Asp300 carbonyl oxygen and the nitrogen atom of His305 Interestingly, this interaction between these two residues has been suggested to mediate the information flow during substrate binding and catalysis [13] The conformation adopted by the loop structure in W58L is another snapshot for different possible conformations that can be adopted by the mobile loop when there is no substrate present
In the crystal structure of the W58A mutant (Fig 5A) no significant deviations in the active site architecture were
Fig 3 Kinetic analysis of a-amylase enzymes (A) Comparison of
action pattern in HSAmy and W58 mutants The enzymes are given in
the order from the top: HSAmy, W58Y, W58L and W58A The
arrows indicate cleavage positions and the numbers reflect the percent
cleavage observed at each point Note that the mutants W58A and
W58L, which do not possess an aromatic residue at position 58, have
distinctly different cleavage pattern than either HSAmy or W58Y (B)
Subsite maps for HSAmy (solid bar) and W58L (open bar) The arrow
indicates the scissile bond The reducing end of maltooligomers is
situated at the right hand of the subsite map Negative energy values
indicate binding between the enzyme and aligned glucopyranosyl
res-idues, while positive values indicate repulsion.
Trang 8observed when compared with either W58L or HSAmy
except for an altered orientation of the His305 side chain
compared with HSAmy/acarbose complex (v2171, W58A/
acarbose vs v2 )114, HSAmy/acarbose complex) This
altered conformation alone could not account for the
significant reduction in the kcatas a mutation of His305 to
Ala reduced the kcatby only 15-fold ([35]; Table 2) While
His305 is known to shift its position in the liganded
structures to interact with the bound oligosaccharide
[11,13], in this structure (W58A complex) part of the loop
(residues 305, 306 and 307) is not well defined This
suggested that the presence of a well-occupied sugar moiety
at subsite)2 might be required for interaction mobilizing
the entire mobile loop
The other notable feature of the W58A/acarbose complex
that is different from the wild-type/acarbose enzyme is the
way acarbose was modified in the crystal Unlike the
wild-type/acarbose complex, wherein acarbose was modified into
a hexasaccharide (PDB code 1mfv), only a
pseudotrisac-charide (acarvosine-glucose) was fully occupied in the
complex W58A/acarbose (Fig 5B) The trisaccharide is
part of the acarbose (acarvosine-glucose) but lacks the
reducing end glucose unit The three sugar rings of the
bound ligand occupy subsites)1, +1 and +2 in a manner
nearly identical to the same trisaccharide component in the
wild-type enzyme [13] The subsites corresponding to the
nonreducing end, subsites )4, )3 and )2 are not fully
occupied The size and shape of the difference density can be
interpreted by fitting the acarvosine-glucose moiety, which
is produced by hydrolytic cleavage of acarbose by the
enzyme in the crystal Alternatively, the observed map could
be due to a longer saccharide formed through a
transgly-cosylation reaction but exhibiting significant positional
disorder at these sites Modeling sugar units at)4, )3 and
)2 subsites resulted in an increase in Rfreeas well as very
high thermal parameters for the sugar atoms (<B>> 60
A˚2 vs <B> of 35 A˚2 for the subsite )1, +1 and +2
atoms) Refinement with partial occupancy for the atoms at
these subsites also did not improve the model Therefore,
only water molecules, which satisfied the criteria given in the
Materials and methods section, were modeled into the
disordered density
The structural analyses reveal that the inhibitor binding
at subsites )1, +1 and +2 has little impact on the
interactions and orientation of the catalytic groups in the
active site The complex W58A/acarbose displayed a secondary sugar-binding site on its surface centered on the residues Trp284 (and Tyr276) This binding site has been previously observed in the complex structures of wild-type HSAmy and the mutant D306 lacking the mobile loop residues 306–310 [13] Smaller oligosaccharides have been observed to occupy similar surface sites in several a-amylases including porcine pancreatic a-amylase [36] and barley a-amylase [37]
Discussion
Enzymatic properties of Trp58 mutants The major effect of the mutation appears to be the loss of the catalytic efficiency as illustrated by the decrease in the
kcatand an increase in Kmfor smaller oligosaccharides This suggested that the transition state stabilization is hampered
by the removal of the bulky side chain in the middle of the binding pocket and that interactions around subsites)2 and )3, which control the substrate binding, might be affected This is partially supported by results from the subsite binding affinity using CNP derivatives The ability of the mutants W58A and W58L to bind the substrates such as G5 and G6 in several binding modes, suggests that there is flexibility of binding around these subsites The crystal structure of the W58A complex provides some evidence for the flexibility in the binding In this structure, clear electron density was visible only for subsites )1, +1 and +2 (Fig 5B) The difference density at the other sites was too weak to fit additional saccharide residues The very poor electron density observed at subsites)4, )3 and )2 suggests that glucose units at these subsites might be highly positionally disordered The positional disorder around subsites)2 and )3 has been suggested as a possible reason for the absence of binding at subsites in the crystal structure
of acarbose-soaked human pancreatic a-amylase mutant D300N [12]
It is known that a-amylases, can display transglycosy-lation activity in the crystal in which the cleavage products are rearranged to form an extended oligosac-charide species Several recent crystallographic studies strongly support that transglycosylation activity occurs in the crystals of a-amylases [38–40] During a transglyco-sylation reaction, the glycosyl covalent intermediate is
Table 3 Action pattern of HSAmy and the W58L mutant.
Substrate Enzyme
Products of hydrolysis (area % of CNP-glycosides) CNP-G 1 CNP-G 2 CNP-G 3 CNP-G 4 CNP-G 5 CNP-G 6
Trang 9attacked by an oligosaccharide moiety instead of water to
lead to a longer sugar chain We have recently shown
that mutants of HSAmy can be used in synthetic
chemistry for producing oligosaccharides by transglyco-sylation [41] Interestingly, while the Trp58 mutant exhibits such an activity in solution (Fig 2B), evidence for such a reaction in the crystal is not very clear due to positional disorder exhibited by the bound pseudooligo-saccharide Thus, although additional density is present
at subsites )4, )3 and )2, only a trisaccharide moiety was modeled in to the active site It is also possible that the added acarbose might have been cleaved to generate
a trisaccharide, which accumulates over the soaking period Due to the flexibility existing in the binding pocket, the concentration of the extended pseudooligo-saccharide is less and hence very low density is observed for such a higher oligosaccharide in the crystal Partial support for this comes from the length of the bound pseudooligosaccharide observed at the secondary binding site in the W58A complex This site also shows clear evidence for only a trisaccharide Thus, in the W58A mutant, the void generated by the absence of Trp at position 58 might be lead to flexibility in the binding of longer oligosaccharides even when they are present Interestingly, the mutants W58L and W58A cleave G3 while HSAmy does not as the number of nonproductive binding modes is reduced in these mutants
Conformational freedom at subsites)2 and )3
in W58A mutant
In the study of barley a-amylase, it was shown recently that Met53 (equivalent to Gln63 at subsite )2 in HSAmy) was required for wild-type kinetic properties such as affinity [42] Inadequate binding at subsite )2 caused distortions at the subsite )1 In the mutants studied here, such a distortion at subsite )1 may not occur as subsite )1 is fully occupied The interactions around this subsite agree well with interactions observed around subsite )1 in wild-type complex [13] However, local rearrangement of some side chain residues around Trp58 does occur, most notably in His305 and Lys352 Two water molecules bridging Asp356 and subsite )2 glucose moiety are also absent (Fig 6) It should be pointed out, however, that at the present resolution (2.1 A˚), the mobile loop His305 side chain is not well resolved This might be taken to be suggestive of the inability of the loop to become ordered upon saccharide binding, a characteristic feature in a-amylases containing such a mobile loop, as critical subsites)2 and )3 are not occupied Nonetheless, from the structural and kinetic data obtained in this study for the W58A/L/Y mutation,
it is clear that the residue Trp58 plays a critical role in the proper binding of the substrates and thus, for maintenance of the optimal catalytic activity of HSAmy
The role of Trp58 in enzyme activity Several conserved residues, dispersed throughout the sequence, are juxtaposed around the active site of a-amylases, some of which have been shown to be important in the enzyme activity [12,13,20,33,35,43,44] The potential role of many of these residues in the hydrolytic activity can be easily surmised from the available crystal structures of a-amylases in complex with acarbose-derived
Fig 4 Structure analysis of the HSAmy mutants (A) Stereodrawing of
the 2Fo-Fc omit maps corresponding to residues 58 and 59 in the
mutant W58L (B) Superposition of the mobile loop of the active site
region in the HSAmy enzymes HSAmy/acarbose complex (thick lines;
PDB Code 1mfv) and W58L (thin lines) Note in the absence of a
bound oligosaccharide, the residue His305, which is part of a mobile
loop, occupies a different space in W58L and lacks the hydrogen bond
between the His305 nitrogen atom and the Asp300 carbonyl oxygen
atom (C) Stereodiagram of the 2Fo-Fc omit map corresponding to the
mobile loop (residues 304–310) Unlike the wild-type structure (PDB
Code 1smd), this region of the structure is well defined In (A) and (C),
the electron density map has been contoured at 1 r and the final
refined coordinates of the corresponding oligosaccharide residues are
overlaid.
Trang 10pseudooligosaccharides The residue Trp58 occurs in a loop
region following the b2 strand of (b/a)8-barrel fold that has
been suggested to be important from the evolutionary point
of view in a-amylase and several other (b/a)8-barrel enzymes
[45] In spite of this importance, the sequence similarity
around b2-a2 loop region is very thin ([18]; http://www
quimica.urv.es/pujadas/AAMY/AAMY_01/; follow the
link Multialignments) The length of this loop varies in size
in different a-amylases and contains one invariant residue
Tyr62 that provides a stacking interaction at subsite)1 An
examination of the reported a-amylase structures
contain-ing acarbose-derived pseudooligosaccharides revealed that
noncontiguous residues occupy the space occupied by
Trp58-Trp59 in HSAmy For example, in TAKA-amylase
[46], residues Arg344 and His80 are located at the positions
occupied by Trp58 and Trp59, respectively) Thus, the
stacking interaction provided by the Trp59 in a-amylases
appears to be compensated by His80 in TAKA-amylase
However, as a result of the variations in the sequence, HSAmy and TAKA-amylases bind pseudooligosaccharides
in two orientations (Fig 7) [13,46] The conformational freedom of the substrate, if any, in TAKA-amylase is restricted probably due to the orientation of two peptide segments TTAYG(72–76) and GDNTV(167–171) around subsite)3 Modeling studies showed that the residues Trp58 (and Trp59) of HSAmy will encounter severe steric inter-actions with the pseudooligosaccharide if the sugar units occupying subsite)2/)3 adopt alternate conformations as observed in TAKA-amylase (Fig 7) Interestingly, the size
of the substrate-binding pocket around the glycone subsites
in a-amylase enzymes that possess Trp58Trp59 segment appears to be larger Why mammalian a-amylases and some other bacterial a-amylases possess a larger substrate-binding pocket is unclear at present Nonetheless, these amylases with Trp58-Trp59 segment also possess a loop segment GHGGA (residues 304–310 in HSAmy and residues 268–
Fig 5 Difference electron density maps (omit maps) in the mutants W58L and W58A/acarbose complex (A) Stereodrawing of the 2Fo-Fc omit maps corresponding to residues 58 and 59 in the mutant W58L (B) Stereodrawing of the 2Fo-Fc omitting density maps corresponding to the bound oligosaccharide in W58A This complex is made up of a trisaccharide and is named according to subsite location The electron density map has been contoured at 1 r and the final refined coordinates of the corresponding oligosaccharide residues are overlaid.