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Oct 19, 2016 - starch on the pancreatic amylase, distinct from the active site of the amylase ... have been with barley α-amylase, a classic member o...
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Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic α‑Amylase Xiaohua Zhang,† Sami Caner,‡ Emily Kwan,† Chunmin Li,‡ Gary D. Brayer,‡ and Stephen G. Withers*,†,‡ †

Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1 Department of Biochemistry and Molecular Biology, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3

‡

S Supporting Information *

ABSTRACT: Starch provides the major source of caloric intake in many diets. Cleavage of starch into malto-oligosaccharides in the gut is catalyzed by pancreatic α-amylase. These oligosaccharides are then further cleaved by gut wall α-glucosidases to release glucose, which is absorbed into the bloodstream. Potential surface binding sites for starch on the pancreatic amylase, distinct from the active site of the amylase, have been identified through X-ray crystallographic analyses. The role of these sites in the degradation of both starch granules and soluble starch was probed by the generation of a series of surface variants modified at each site to disrupt binding. Kinetic analysis of the binding and/or cleavage of substrates ranging from simple maltotriosides to soluble starch and insoluble starch granules has allowed evaluation of the potential role of each such surface site. In this way, two key surface binding sites, on the same face as the active site, are identified. One site, containing a pair of aromatic residues, is responsible for attachment to starch granules, while a second site featuring a tryptophan residue around which a malto-oligosaccharide wraps is shown to heavily influence soluble starch binding and hydrolysis. These studies provide insights into the mechanisms by which enzymes tackle the degradation of largely insoluble polymers and also present some new approaches to the interrogation of the binding sites involved.

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amylases. The most complete studies of amylase SBSs to date have been with barley α-amylase, a classic member of CAZy family GH13 for which two principal SBSs have been identified.7−9 One of these seems to play a role in binding to and hydrolyzing soluble starches, while the other functions with insoluble starch granules. These insights were obtained through X-ray crystallographic analyses in conjunction with kinetic and binding studies, both with the wild-type enzyme and with variants modified within individual SBSs. A challenge for such studies is that the binding affinities determined are not necessarily truly reflective of the individual SBS, but rather of some combination of binding at the SBS and at the active site; thus, these are typically maximal estimates of affinity. Human pancreatic α-amylase (HPA) is a GH13 endoglycosidase that is responsible for the degradation of starch to oligosaccharides within the gut. These oligosaccharides are then hydrolyzed to glucose by α-glucosidases on the gut wall. Detailed structural and kinetic studies of HPA with soluble substrates have provided valuable insights into the structure and chemical mechanism of this key digestive enzyme.10−13 However, little is known about its action on insoluble starch

nzymes that degrade polysaccharides often contain binding sites remote from the active site to assist in the cleavage of substrates. Various specific roles for these binding sites have been suggested.1−3 The most probable is a role in localization of the enzyme on the polymeric substrate while allowing the active site to effect catalysis multiple times without release from the substrate. Other suggested roles include disruption of substrate structure, guidance of the polymer chain into the active site, and allosteric regulation. These remote binding sites are often located within independently folded binding domains attached to the catalytic domain through a polypeptide linker; indeed, some polysaccharide hydrolases contain several such carbohydrate binding modules. The structures and roles of these modules have been extensively reviewed.4−6 Extensive data and a sequence-based classification can be found in http://www. cazy.org/Carbohydrate-Binding-Modules.html. A less well studied class of binding sites is that found on the surface of the catalytic domain rather than as independent domains, and these are termed surface binding sites (SBSs).1−3 Because they cannot typically be produced independently, it is more difficult to experimentally separate the roles of these SBSs from those of the active site and thereby assess their contributions to catalysis. SBSs have so far been found predominantly on enzymes that degrade starch and closely related polymers, particularly on © XXXX American Chemical Society

Received: September 28, 2016 Revised: October 17, 2016

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DOI: 10.1021/acs.biochem.6b00992 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry granules and the possible presence and role of SBSs, though one such preliminary study has been published on the closely homologous salivary α-amylase.14 While the human diet has primarily included solubilized starches since the conception of cooking, HPA originally evolved to degrade raw starch and has likely changed little since, as evidenced by its high degree of homology with that of our porcine cousins.15,16 In this report, we identify a number of candidate SBSs on the surface of HPA on the basis of crystallographically observed non-active site binding modes for sugars. We then describe the design, construction, and analysis of variants in which binding at each of these potential SBSs, individually, is disrupted. Studies with both large and small soluble substrates, as well as starch granules, performed in the absence and presence of inhibitors that block the HPA active site, both reversibly and irreversibly, serve to identify the contributions of key SBSs.

A selected colony was used to inoculate 60 mL of BMGY (buffered glycerol) medium in a baffled flask, and this colony was grown at 30 °C overnight. Ten milliliters of cell culture was transferred to an additional 600 mL of BMGY medium and grown in a 2 L baffled flask at 30 °C for 1 day. The cells were collected at 5000 rpm and 4 °C for 15 min and resuspended in 200 mL of BMMY (buffered methanol) medium. Two milliliters of 50% (v/v) methanol in water was added twice a day to the cell culture, and the culture continued to grow at 30 °C for 2 days. The cells were harvested at 6000 rpm and 4 °C for 20 min, and the supernatant was filtered through a glass microfiber filter GF/C (Whatman, catalog no. 1822125), followed by concentration with an Omega 76 mm 10K membrane (Pall Filtron, catalog no. MO001076) to ∼20 mL. The volume of the supernatant was adjusted with loading buffer [50 mM sodium chloride and 100 mM potassium phosphate (pH 7.5)] and loaded onto the Phenyl-Sepharose CL-4B column. The column was washed with additional loading buffer (5× bed volume) followed by elution with deionized water. All the fractions were checked on sodium dodecyl sulfate− polyacrylamide gel electrophoresis, and positive fractions were pooled and concentrated to 4−8 mL using an Amicon Ultra-15 centrifugal filter (Millipore, 30K). Concentrated buffer was added to this deep green solution to yield final buffer concentrations of 20 mM potassium phosphate and 25 mM sodium chloride (pH 7.0). Endoglycosidase F fusion protein [1:100 (w/w)] was added for deglycosylation, and the mixture was left at room temperature overnight. The solution was then passed through a HiTrap Q Sepharose Fast Flow anion exchange column. The flow-through, which contained the HPA variant protein, was concentrated by an Amicon 30K filter. Enzyme concentrations were determined spectrometrically using a Varian Cary 300 UV/vis spectrometer and with an A0.1% of 2.24 at 280 nm for both wild-type and variant HPA. Kinetic Studies with CNP-G3. 2-Chloro-4-nitrophenyl αmaltotrioside (CNP-G3) was purchased from Genzyme Inc. (Cambridge, MA). Michaelis−Menten parameters of HPA variants for CNP-G3 were determined by monitoring the increase in absorbance at 400 nm using a Varian CARY 300 spectrophotometer. Quartz cuvettes (200 μL) with a path length of 1 cm were used. The concentration of enzyme used was approximately 20−30 nM (depending on enzyme activity) for all assays. Initial rates were measured using six to eight different CNP-G3 concentrations ranging from 1 to 20 mM in HPA buffer [50 mM sodium phosphate and 100 mM sodium chloride (pH 7.0)] at 30 °C and fit to the Michaelis−Menten equation by nonlinear regression using GraFit 7.0.0 (Erithacus Software, London, U.K.) to obtain Vmax (kcat) and KM. Hydrolysis of Soluble Starch. Determination of the rate of enzymatic hydrolysis of soluble starch was performed by quantifying the concentrations of sugar reducing ends using the dinitrosalicylic acid (DNS) assay.19 A stock soluble potato starch (Sigma-Aldrich, catalog no. S-5651) solution (10 mg/ mL) was made in HPA buffer [50 mM sodium phosphate and 100 mM sodium chloride (pH 7.0)] and boiled gently until all the solids were dissolved. Starch solutions at various concentrations (0−6 mg/mL) were prepared by diluting the stock solution with HPA buffer. Purified enzyme (final concentration of 3−8 nM depending on enzyme activity) was then added to 4 mL of a diluted starch solution and the mixture incubated for an appropriate length of time at 30 °C. At different time points, 500 μL of the mixture was withdrawn and the reaction quenched immediately by addition of an equal

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MATERIALS AND METHODS Site-Directed Mutagenesis. All commercially available vectors, restriction enzymes, T4 DNA ligase, fast alkaline phosphatase, and Pichia pastoris strains were acquired from ThermoFisher Scientific (Waltham, MA). All chromatographic resins were purchased from GE Healthcare (Little Chalfont, U.K.). Site-directed mutagenesis using overlap extension polymerase chain reactions (OE-PCR) was performed as described by Higuchi.17 Two flanking primers (primers A and D) on either end of the mutant sequence and two internal primers (primers B and C) that contain the mismatched bases and hybridize at the mutation region were used. In the first round, two separate PCRs were used to create the AB and CD oligos from the pPic9-HPA template with two universal primers 5′ AOX1 and 3′ AOX1 (as primers A and D for all the mutants). The oligonucleotide sequences of forward and reverse primers for each variant are listed in the Table S1. Each PCR for AB and CD fragments was performed in a total volume of 50 μL containing the template (10 ng), the pair of primers (20 pmol), each of the four deoxynucleotides (20 pmol), and Pf u polymerase (1.25 units) in 1× Phusion HF buffer (Fermentas). The reaction was repeated 25 times by cycling the temperature of the reaction mixture using a GeneAmp PCR system 2400 (PerkinElmer), with temperature cycles of 98 °C for 10 s, 56 °C for 30 s, and 72 °C for 35 s. Both AB and CD fragments were purified with a QIAquick gel extraction kit (Qiagen) and then hybridized in a subsequent “fusion” reaction with the aid of primers A and D. PCR conditions were determined as described above with slight differences. (1) Both AB and CD fragments (20 ng) were used as templates, and (2) the temperature cycle was modified to 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 50 s. The resulting AD fragment was gel purified and digested with restriction enzymes SacI and NotI in 1× FastDigest buffer at 37 °C for 1 h and subcloned into the pPic9K vector after purification by a QIAquick PCR kit (Qiagen). Each mutant plasmid was then transformed into TOP10 Escherichia coli. The resulting colonies were inoculated in starter cultures (5 mL). Plasmid DNA was purified using the QIAprep Spin Miniprep purification kit (Qiagen). cDNA sequencing was performed to verify the point mutation at the Nucleic Acid and Protein Service Unit at the University of British Columbia, and the sequence was then linearized using SacI and transformed into P. pastoris strain GS115 by electroporation. Protein Expression. HPA and its variants were produced in P. pastoris and purified as previously described by Rydberg.18 B

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substrate and inhibitor complexes where additional carbohydrate moieties were resolved outside the catalytic center of this enzyme. In addition, the structure of the enzymatically compromised HPA D300N−G8 (maltooctaose) complex was completed herein to add further to this complement of SBSs. This complex was formed by first growing crystals of the D300N variant HPA enzyme as previously described for the wild-type enzyme,10,11 followed by soaking in a 200 mM solution of G8 overnight and then infusing a further aliquot of G8 just before freezing the complexed crystal in liquid nitrogen for use in X-ray diffraction analyses. Crystallographic data were collected at cryogenic temperature (100 K) using an ADSC Quantum detector at the Stanford Synchrotron Radiation Lightsource (Stanford, CA). The obtained diffraction data were indexed and integrated using XDS.24 Some reflections were excluded to remove ice rings from the final data. Therefore, data were truncated according to an overall data completeness criterion of around 90%. The structure of the HPA D300N−G8 complex was determined by molecular replacement employing PHASER25 by using the high-resolution wild-type HPA structure as a starting model23 [Protein Data Bank (PDB) entry 4X9Y]. Structure refinement of this complex was accomplished using Phenix.26 The refinement options included coordinate and isotropic atomic displacement parameters as well as atomic occupancies for ligands in the model. Furthermore, each ligand was defined as a constrained group in the occupancy refinement. As a result, the occupancies of all ligands were determined (maltose at 91%, tetraose 1 at 81%, tetraose 2 at 96%, hexaose 1 at 94%, and hexaose 2 at 94%). To confirm binding of malto-oligosaccharides of variable lengths, a simulated annealing omit map of each identified ligand binding site was calculated. The resulting electron density map was inspected at the 2σ or 3σ level and confirmed ligand binding in each case. Where necessary, the obtained model was optimized by iterative model building using the molecular visualization software COOT.27 Water molecules were automatically added to the model through the refinement process and were evaluated manually on the basis of hydrogen bonding potential with protein and ligand atoms. Backbone dihedral angles in the HPA D300N−G8 complex were distributed within the most favored (96.6%) and additionally allowed regions (3.2%) of the Ramachandran map. The outlier (Thr163) was inspected and judged to be real according to its well-ordered surrounding electron density. Additionally, Thr163 forms two hydrogen bonds that contribute to binding of the active site-bound hexaose. Data collection and structural analyses statistics can be found in Table 1.

volume of a DNS solution (10 mg/mL 3,5-dinitrosalicyclic acid and 300 mg/mL sodium potassium tartrate in a 0.4 M sodium hydroxide solution). The color was developed by boiling the stopped samples for 5 min, followed by cooling to room temperature. The absorbance was measured spectrophotometrically at 540 nm and plotted versus reaction time. Extrapolating the curve gave a straight line with the slope as the initial rate of starch hydrolysis. A glucose standard curve was used to calibrate the readings. The rates were plotted against substrate concentration and fit to a Michaelis−Menten equation in GraFit 7.0.0 to obtain Vmax (kcat) and KM. Starch Granule Adsorption Assay. The adsorption of purified enzymes to starch granules was assayed according to a procedure that is slightly modified from that previously reported, and using the NanoOrange protein quantitation kit from Invitrogen (Carlsbad, CA).20 Potato starch was first washed three times in deionized water and twice in ethanol and air-dried.21 Suspensions of this material at concentrations of 0− 200 mg/mL in HPA buffer were prepared and cooled at 4 °C for at least 10 min. Recombinant HPA variants (final concentration of around 100 nM) were incubated with the prechilled starch suspensions (1 mL) on a rotating frame at 4 °C for 60 min. The mixtures were then centrifuged (4 °C for 10 min) to separate all the starch, along with bound amylase, from free enzyme; 500 μL of the resulting supernatant was concentrated to a volume of approximately 10 μL using an Amicon Ultra-15 centrifugal filter 30K (Millipore, Billerica, MA). The concentrated enzyme solution was diluted with a 1× NanoOrange working solution to achieve a final volume of 1 mL. The samples were heated in the dark at 95 °C for 10 min. After being cooled to room temperature, the solution was transferred into a full-volume methacrylate cuvette (Fisherbrand), and the fluorescence of the unbound enzyme was measured at 590 nm (excitation at 485 nm). The activity (expressed as the percentage of bound proteins when compared with a no-starch control) was plotted against the starch concentrations, and the data were fitted to a one-site binding model using GraFit 7.0.0. b=

Bmax [S] [S] + Kd

where b is the bound enzyme fraction, [S] the starch concentration, and Bmax the maximal fraction of enzyme bound. Modified Adsorption Protocol Including Montbretin (MbA). HPA and its surface variants (20 μM) were incubated with MbA (400 μM) in HPA buffer at room temperature for 1 h prior to the addition (5 μL) of the enzyme complex to prechilled starch suspensions (1 mL). The adsorption assay followed the protocol of the general adsorption assay. Modified Adsorption Protocol with Inactivated HPA. In situ elongation of epi-cyclophellitol and inactivation of HPA were accomplished by preincubating HPA (1.6 nmol) with 4′O-methyl α-maltotriosyl fluoride13,22 (28 μmol) and epicyclophellitol23 (28 μmol) in 200 μL of HPA buffer at 30 °C, until no residual enzyme activity could be detected. The excess elongation reagents were removed by use of an Amicon 30K spin filter, and the inactivated enzyme (0.1 nmol) was added to the prechilled starch suspensions (1 mL), followed by the general adsorption assay. X-ray Crystallography: Crystal Growth, Data Collection, and Structure Determination. HPA SBSs were inventoried by reference to previously determined HPA

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RESULTS AND DISCUSSION Crystallographic studies performed on HPA in which oligosaccharides or oligosaccharide precursors were soaked into crystals of HPA have in many cases identified binding modes for these ligands outside the active site (Table 2). Because these soaking experiments are typically performed using high ligand concentrations (∼50−200 mM), it is not clear whether the binding modes so identified have any biochemical or physiological relevance. In a similar vein, it is quite possible that other “true” binding modes have been missed if they are occluded by crystal lattice packing interactions. Nevertheless, these observed remote binding modes provide an excellent starting point for potential identification and validation of SBSs C

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Biochemistry Table 1. Diffraction Data Collection and Refinement Statistics for the HPA D300N−G8 Complexa wavelength (Å) resolution range (Å) space group unit cell total no. of reflections no. of unique reflections multiplicity completeness (%) mean I/σ(I) Wilson B factor Rmerge CC1/2 Rwork Rfree no. of non-hydrogen atoms macromolecules ligands/ions waters no. of protein residues root-mean-square deviation for bonds (Å) root-mean-square deviation for angles (deg) Ramachandran plot (%) favored allowed outliers average B factor (Å2) macromolecules ligands and ions waters a

Table 2. Observed Binding Interactions of Sugar Analogues and Oligosaccharides at Sites Outside the Active Site on the Surface of HPAa

0.976067 38.46−2.30 (2.36−2.30) P212121 52.2 Å, 73.6 Å, 135.4 Å, 90.0°, 90.0°, 90.0° 93522 (7633) 21229 (1715) 4.4 (4.5) 88.9 (99.7) 24.23 (8.13) 24.80 0.04789 (0.1888) 0.999 (0.975) 0.1511 0.1886 4352 3946 247/2 157 496 0.009

surface binding site

ligands

3A

all complexesb−e at the terminus of the active site binding cleft, acts as a blocker 5FIdoF and maltoseb,c

3B

maltohexaosec

4

maltotetraosec

5 6 7

5FIdoFb 5FIdoFb maltotetraosec

8

maltotetraose and MeG2Fc,d MeG2−5FIdod NAGe

1 2

9 10

amino acid residues involved

mutation(s)

active site cleft N152

ND N152W

K140, T155, D159, W203, G205, D206, E171, R176 W134, Y174, S132, K172, D135 forms a WY clamp adjacent to SBS3A N363, E369, R20, V366, K368 R80, T84, N220 N250 K261, E272, Y276, N279, W284 forms a WY clamp T376, W388, R392, R389, K322, Q390 D433, W434 N459, N461A, K466

W203A

E369R R80Af N250R Y276A, W284A W388Af W434A N461A

a

HPA structures for which ligands outside the active site were determined, along with the respective PDB entry designations and references. b5FIdoF is 5-fluoro-β-L-idosyl fluoride (PDB entries 3IJ8 and 3IJ913). cMaltose, maltopentaose, maltotetraose [PDB entry 5TD4 (this work)]. dMeG2F is 4′-O-methyl-α-maltosyl fluoride (PDB entry 3IJ713). eNAG is N-acetyl-D-glucosamine (PDB entries 1U30 and 4GQQ22). fVariant unstable.

1.109

96.6 3.2 0.2 22.90 22.83 40.06 25.07

Statistics for the highest-resolution shell are given in parentheses.

on HPA. Figure 1 presents a composite of ligand binding modes observed at distinct surface sites on HPA. The PDB entries for each structure, from which these binding modes have been extracted, are provided in the footnotes of Table 2. The active site is designated site 1 and is where the catalytic nucleophile Asp197 and the acid/base catalyst Glu233 are located, within subsite −1. The active site requires five subsites to be filled for efficient cleavage of malto-oligosaccharides (−3 to +2).11 Other observed potential SBSs can be divided into two groups: those that contain oligosaccharides of three or more sugars (maltotriose) and those that contain a monosaccharide or a disaccharide. This distinction is made on the basis of the fact that the binding of longer oligosaccharides is far more likely to signify a site that normally binds to a polymeric structure. Some caution is also warranted in these studies given that the monosaccharide observed bound in SBS 3A, 5, and 6 is a difluorinated derivative with an inverted configuration at C-5 relative to glucose, making it a less than perfect mimic.13 However, recognizing that crystal lattice restraints might limit binding modes and given that this sugar contains a vicinal triol of the same configuration as glucose, we still examined these sites, even though they were considered as less likely candidates. Table 2 provides a summary of the putative SBSs and ligands structurally identified on the surface of HPA, along with the amino acid residues found to be in contact with ligands. As seen

Figure 1. Composite of snapshots of observed mono-oligosaccharide and oligosaccharide ligand binding sites on the surface of HPA. Site 1 is located in the active site cleft, and sites 2−10 are remote from this region and may have the potential to bind elements of starch granules. Site numbering corresponds to the list in Table 2 of interactive residues found in individual binding sites. Note that putative SBS sites were identified from a series of HPA structures and were not seen to be fully occupied at one time. The Protein Data Bank entries for the structures of complexed HPA examined are listed in the footnotes of Table 2.

in other studies of carbohydrate binding sites on proteins, both hydrogen bonding and hydrophobic interactions contribute to binding interactions, with the latter being frequently mediated through Tyr and Trp residues. This is particularly evident in SBS 7, where Tyr276 and Trp284 together create a hydrophobic platform that matches the lower faces of the sugar rings in the bound structure (Figure 2). Indeed, this configuration is highly reminiscent of that seen in other D

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site. Kinetic parameters equal or close to those of the wild-type enzyme indicate that the variant is correctly folded. Indeed, all variants show kcat/KM values within 3-fold of the wild-type value, indicating that they had all folded into the catalytically active form. In addition, the fact that no large differences were observed confirms expectations that remote binding of an oligosaccharide is unlikely to affect active site performance significantly; thus, allosteric effects are not evident. Surprisingly, for most of the variants, hydrolysis of soluble starch was not greatly compromised, with kcat/KM values for all except Trp203Ala being within a factor of 3 of the wild-type value (Table 3). The >6-fold reduction in kcat/KM for the Trp203Ala variant suggests important interactions of SBS 3 with the soluble starch. This is the site that has a circular surface depression that could accommodate a helical oligosaccharide conformation and is thus a likely candidate for interaction with soluble starch chain ends. Its proximity to the active site (12 Å away) lends further credibility to this notion. Binding to Starch Granules. A commonly employed assay for the binding of enzymes to starch granules is one in which a well-washed potato starch suspension, chilled at 4 °C, is incubated with defined quantities of the protein of interest for 1 h and then centrifuged. The expectation in this approach is that incubation at low temperatures will temporarily halt hydrolysis. The precipitate thus obtained contains the starch granules with any adsorbed protein, while the supernatant contains the unbound protein. Measurement of the amount of free protein in the supernatant allows calculation of the bound fraction. Repeating such measurements at a series of starch concentrations allows the dissociation constant to be determined by fitting the data to a Langmuir adsorption isotherm (one-site binding model).

Figure 2. Individual views of key SBS binding sites on HPA with bound ligands: (a) active site and SBS 2, (b) SBS 3A/3B, and (c) SBS 7. Sugar analogues are shown as purple sticks. Residues of wild-type HPA are colored green, and the folded structure of HPA bound with ligands is colored green. The calcium ion bound to HPA is represented as a red sphere.

proteins that bind to insoluble polysaccharides, such as the cellulose binding domains4,28,29 and the polysaccharide monooxygenases that are responsible for oxidative cleavage of cellulose, starch, and chitin.30−32 Furthermore, Trp203 plays an important role in SBS 3A, wherein it serves as a central “axle” around which a circular surface depression exists. A similar general binding mode has been seen in other GH13 enzymes, such as cyclodextrin glucanotransferase, where a tyrosine residue plays an equivalent role.33 Immediately adjacent to SBS 3A is the SBS 3B site, which consists of Trp134 and Tyr174, and like SBS 7, a ligand is found threaded through its hydrophobic contact region. The mutational strategy chosen to disrupt binding at each SBS was generally to replace key tryptophans or tyrosines with alanine, thereby removing the stabilizing hydrophobic and hydrogen bonding interactions at that locale. Other positions were typically substituted with arginine to introduce a bulky hydrophilic residue or with alanine to remove hydrogen bonding (Table 2). Eight of the 10 variants expressed well and provided soluble active enzymes. Unfortunately, despite repeated attempts, variants Arg80Ala and Trp388Ala could not be stably expressed; thus, SBS 5 and 8 could not be evaluated. Evaluation of Kinetic Parameters. Kinetic parameters for hydrolysis of the oligosaccharide substrate CNP-G3 were first determined for each variant protein and wild-type HPA and are listed in Table 3. This substrate binds only in the active site, with its maltotriosyl moiety in subsites −1, −2, and −3, and is cleaved to release maltotriose and 2-chloro-4-nitrophenol, which is readily monitored by UV−vis spectrophotometry. The wild-type HPA kinetic parameters determined with this substrate are therefore a reflection of the integrity of the active

b=

Bmax [S] [S] + Kd

To conduct such studies, a sensitive method for determination of protein concentrations was required. Activity assays were considered unreliable in this case because any residual starch would compete with CNP-G3 for binding in the active site, lowering the activities and thereby underestimating protein concentrations. Use of NanoOrange dye proved to be the most satisfactory approach because its large fluorescence increase upon interaction with proteins allowed measurements of protein concentrations down to 10 ng/mL. Results from these studies are listed in Table 4 and shown graphically in Figure 3. Surprisingly, once again, the apparent Kd values for most variants were very similar to those of the wild-type enzyme except for those of Asp197Asn, for which the Kd value

Table 3. Catalytic Efficiencies of HPA Variants for Hydrolysis of CNP-G3 and Soluble Starch CNP-G3 hydrolysis protein wild-type HPA N152W W203A E369R N250R Y276A W284A W434A N461A

−1

kcat (s )

KM (×10

± ± ± ± ± ± ± ± ±

3.2 2.2 4.0 3.8 2.7 3.1 2.9 3.6 2.1

20.8 14.7 8.7 10.5 7.7 17.9 16.1 17.7 9.9

0.5 0.3 0. 1 0.6 0.5 0.5 0.2 0.2 0.6

± ± ± ± ± ± ± ± ±

−3

M)

0.3 0.2 0.2 0.7 0.5 0.3 0.1 0.2 0.4

soluble starch hydrolysis −3

kcat/KM (×10 6.5 6.6 2.2 2.8 2.9 5.7 5.6 4.9 4.8

± ± ± ± ± ± ± ± ±

M

−1

0.6 0.5 0.1 0.6 0.6 0.6 0.3 0.3 1.0

−1

s )

3

kcat (×10 s ) 2.1 1.0 0.6 1.7 0.7 1.9 1.5 1.6 1.7

E

−1

± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0. 1

KM (mg/mL) 0.7 0.6 1.1 0.6 0.3 0.8 0.9 1.2 0.6

± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.3 0.1

kcat/KM (×103 mL mg−1 s−1) 3.1 1.8 0.5 3.1 2.3 2.3 1.7 1.3 3.0

± ± ± ± ± ± ± ± ±

0.2 0.3 0.1 0.5 0.6 0.5 0.2 0.4 0.5

DOI: 10.1021/acs.biochem.6b00992 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry Table 4. Apparent Binding Constants for Binding of HPA Variants to Starch Granules starch granule binding surface binding site 1

2 3 4 6 7 9 10

protein wild-type HPA D197N N152W W203A E369R N250R Y276A W284A W434A N461A

apparent Kd (mg/mL)

Kd (with MbA) (mg/mL)a

Kd (with MeG2ECP) (mg/mL)b

5.9 ± 1.3

0.4 ± 0.1

0.5 ± 0.1

1.0 6.0 7.2 5.3 6.1 16.7 29.1 4.6 3.9

± ± ± ± ± ± ± ± ±

0.1 0.9 1.2 0.9 1.4 2.6 6.7 0.6 0.8

1.0 0.4 1.2 0.6 0.7 5.9 22.3 0.7 1.0

± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.2 0.6 2.9 0.1 0.1

−c 2.6 0.6 0.9 1.4 1.7 4.7 0.4 1.5

± ± ± ± ± ± ± ±

0.3 0.1 0.1 0.3 0.5 0.7 0.1 0.1

Figure 4. Reducing sugar production in the supernatant of the starch suspension, when no enzyme was added (○), when only wild-type HPA was incubated (◇), when HPA was pretreated with MbA (△), and when HPA was pretreated with MeG2-ECP (▽) at 4 °C.

a The values were determined in the presence of MbA. bThe values were determined using MeG2-ECP-treated enzymes. cD197N cannot react with MeG2-ECP.

observed. Two possible solutions to the issue of substrate cleavage were considered. One was to convert all the SBS mutants into double mutants that also contain Asp197Asn and remeasure Kd values. This approach would be extremely timeconsuming. Alternatively, a more efficient solution involves the remeasurement of the Kd values in the presence of a potent competitive inhibitor that binds at the active site. This would have the added benefit of precluding granule binding at the active site, thus ensuring that Kd values determined reflect only SBS binding. Fortunately, we have recently published the discovery and structural analysis of just such an inhibitor, Montbretin A (MbA), which binds to the active site with a Ki value of 8.1 nM.34 This flavonol glycoside (molecular weight of 1229) largely fills the active site (Figure 5a) but binds at no other locales on the surface of HPA.35 Indeed, incubation of wild-type HPA with 20 μM MbA for 1 h prior to the addition of the starch suspension yielded a Kd value for starch granule binding that was >15 times lower than that determined in the absence of MbA (Table 4), clearly supporting the hypothesis that release of oligosaccharides as competitive ligands had indeed resulted in serious underestimates of starch granule affinity for

was much lower, and Tyr276Ala and Trp284Ala, for which Kd values were both much higher, indicating an important role for SBS 7 in starch granule binding. The apparent tighter binding of the active site mutant Asp197Asn compared to that of the wild type was surprising, because such mutations do not usually increase affinity and because tighter granule binding at the active site seems less probable. The other possible explanation is based upon the fact that this variant, unlike all the others, is catalytically inactive. Possibly, the residual activity of the other variants at 4 °C is sufficient to degrade the starch granules to some extent, releasing malto-oligosaccharides that then compete with the granule for binding. If this is the case, then all the Kd values for starch granule binding in Table 4 (except that for Asp197Asn) are underestimates of the true binding affinity. The same would be true of all Kd determinations made by other investigators using this protocol in the literature. To test this possibility, cleavage of starch granules at 4 °C was monitored by measuring reducing sugar release as shown in Figure 4, and indeed, significant hydrolytic activity was

Figure 3. Histogram of relative Kd apparent values for binding of starch to human pancreatic α-amylase and variants. Values are normalized to a Kd of 1 for the wild-type enzyme. F

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the experiment was repeated using a mechanism-based covalent inactivator to permanently block the active site. Recently, we have described the development of α-1,4-glucosyl epi-cyclophellitol as a covalent, active site-directed inhibitor of HPA.23 While this inhibitor provided useful insights into active site structure, it inactivated the enzyme very slowly and its preparation was quite demanding, thus limiting its utility. In search of a simpler approach to covalent inactivation, we explored the ability of HPA to synthesize its own inactivator from the same epi-cyclophellitol “warhead” and then inactivate itself, much as we had done previously with the 5-fluoro sugar inhibitors.13 To that end, we incubated HPA (1.6 nmol) with epi-cyclophellitol (28 μmol) and 4′-O-methyl-α-maltosyl fluoride (28 μmol) as a nonpolymerizing glycosyl donor and monitored HPA activity as a function of time (Scheme 1). After 4 h, the activity had dropped to zero and ESI-MS analysis revealed an increase in the mass of HPA from 56068 to 56582 Da (Figure S1). The mass difference measured at 514 Da (±1) is that expected from the covalent attachment of a single pseudotrisaccharide (m/z 514.5) at the active site. Modeling of its binding mode in the active site, based upon our X-ray structure of the glucosyl epi-cyclophellitol-inactivated structure (PDB entry 5EMY), revealed a complex that occupies much less of the active site than MbA does (Figure 5b). Determination of Kd values for binding of a starch granule to this inhibited enzyme revealed values that are quite similar to those determined in the presence of MbA (Table 4), especially in terms of their trends (Figure 3). Kd values for variants in SBS 7 were not as high as those seen with the MbA complex, possibly suggesting some binding in the (+) sites of the active site in this case. In addition, binding to the Asn152Trp variant was also somewhat compromised relative to what had been seen with MbA. Reasons for this are not obvious, but it may reflect a localized conformational change in the presence of MbA that packs the inserted tryptophan closer to the HPA surface, thereby minimizing disruptive interactions with the starch granule.

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CONCLUSIONS Binding of HPA to starch granules appears to be mediated primarily by SBS 7, a classic polysaccharide binding site with interactions mediated via Tyr276 and Trp284. Interaction with soluble starch, by contrast, appears to occur primarily at SBS 3A and appears to involve sequestration of helical turns of the starch polymer around a tryptophan axis (Trp203). Another complementary driver of starch digestion may be SBS 3B (Trp134 and Tyr174) given its proximity to the SBS 3A site. Interestingly, all of these sites are on the same face of the HPA molecule as the active site; thus, a model in which HPA is anchored to the surface of the granule via SBS 7 is easily envisaged (Figures 2 and 6). Binding in such a way would allow loose chain ends to engage both with the active site groove, with which it is nicely aligned, and with SBS 3A/B. Interactions at the other sites, with the possible exception of SBS 2, which is located to one end of the active site substrate binding cleft, in any case, were not very significant. Indeed, these other sites are quite possibly artifacts of their initial method of identification through crystallographic soaking studies at high ligand concentrations. Studies with barley α-amylase arrive at related conclusions, with a starch granule binding site containing two adjacent tryptophan residues (Trp278 and Trp279) seeming to be important,8 and located in the same general region as SBS 7 in

Figure 5. View of the HPA active site bound with (a) MbA from a structure determined with PDB entry 4W93 and (b) MeG2-ECP [modeled conformation based on the structural determinations of the HPA active site-bound 5FIdoF-HPA (PDB entry 3IJ7) and G-ECPHPA (PDB entry 5EMY)]. Residue Asn152, the residue that was mutated to Trp to block binding at the entrance to the substrate binding cleft, is colored blue.

active enzymes. Confirmation that addition of MbA was inhibiting starch hydrolysis at 4 °C is shown in Figure 4. In light of this finding, Kd values for starch granule binding were thus redetermined for each variant after preincubation with MbA, and the results are listed in Table 4 and shown as relative Kd values in Figure 3. As one can clearly see, the Kd values for each variant decreased substantially upon addition of MbA. All variants, with the exception of those in SBS 7, bind starch granules with a Kd value within a factor of ∼3 of that of the wild-type enzyme. However, binding of the Tyr276Ala and Trp284Ala variants is 15- and 60-fold weaker, respectively, signifying again an important role for SBS 7 in starch granule binding. To ensure that the binding of MbA did not in some way modify binding, or perhaps still allow some substrate turnover, G

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Scheme 1. Proposed in Situ Elongation−Trapping Mechanism for HPA Using Epi-cyclophellitol as the Acceptor and MeG2F as the Donor

Figure 6. Structural overlap of the course of the polypeptide chain of HPA (PDB entry 1HNY, green) and barley α-amylase AMY1 (PDB entry 1HT6, orange). The catalytic nucleophile Asp197 and the aromatic amino acid residues at SBS 7 (Tyr276 and Trp284) of HPA are colored cyan, and the equivalent “starch granule binding site” of AMY1 (Trp278 and Trp279) is colored magenta. These individual residues are represented as stick figures in the enlarged views and individually denoted with prefixes HPA and BAR.

HPA (Figure 6). The two aromatic residues in this region identified as being important in the barley enzyme are colored magenta, while the two identified as being important in HPA are colored cyan. Likewise, crystallographic studies with porcine pancreatic α-amylase also identified Trp284 as a secondary binding site,36 while binding studies of the double mutant (Tyr276Ala/Trp284Ala) of human salivary α-amylase suggested some involvement in granule binding.14 However, results of binding studies in the cases mentioned above are clouded by both the underestimation of glycan affinities in

those cases due to use of active enzymes that release oligosaccharides and the possibility of binding at both the SBS and the active site. Nonetheless, a picture in which attachment of amylases such as HPA to the starch granule surface is mediated through a binding site containing aromatic residues is emerging . The cofacial location of this site with the active site allows chain ends to enter the active site and be cleaved. A second binding site (SBS 3A/B) that accommodates long soluble chains to bind and be digested may well also assist the binding of granules with long “soluble” glycan chains. H

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(7) Robert, X., Haser, R., Gottschalk, T. E., Ratajczak, F., Driguez, H., Svensson, B., and Aghajari, N. (2003) The structure of barley alphaamylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs. Structure 11, 973−984. (8) Nielsen, M. M., Bozonnet, S., Seo, E. S., Motyan, J. A., Andersen, J. M., Dilokpimol, A., Abou Hachem, M., Gyemant, G., Naested, H., Kandra, L., Sigurskjold, B. W., and Svensson, B. (2009) Two secondary carbohydrate binding sites on the surface of barley alphaamylase 1 have distinct functions and display synergy in hydrolysis of starch granules. Biochemistry 48, 7686−7697. (9) Nielsen, J. W., Kramhoft, B., Bozonnet, S., Abou Hachem, M., Stipp, S. L. S., Svensson, B., and Willemoes, M. (2012) Degradation of the starch components amylopectin and amylose by barley alphaamylase 1: Role of surface binding site 2. Arch. Biochem. Biophys. 528, 1−6. (10) Brayer, G. D., Luo, Y., and Withers, S. G. (1995) The structure of human pancreatic alpha-amylase at 1.8 A resolution and comparisons with related enzymes. Protein Sci. 4, 1730−1742. (11) Brayer, G. D., Sidhu, G., Maurus, R., Rydberg, E. H., Braun, C., Wang, Y. L., Nguyen, N. T., Overall, C. H., and Withers, S. G. (2000) Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques. Biochemistry 39, 4778−4791. (12) Rydberg, E. H., Li, C. M., Maurus, R., Overall, C. M., Brayer, G. D., and Withers, S. G. (2002) Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids. Biochemistry 41, 4492− 4502. (13) Zhang, R., Li, C., Williams, L. K., Rempel, B. P., Brayer, G. D., and Withers, S. G. (2009) Directed ″in situ″ inhibitor elongation as a strategy to structurally characterize the covalent glycosyl-enzyme intermediate of human pancreatic alpha-amylase. Biochemistry 48, 10752−10764. (14) Ragunath, C., Manuel, S. G. A., Venkataraman, V., Sait, H. B. R., Kasinathan, C., and Ramasubbu, N. (2008) Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-amylase in substrate hydrolysis and bacterial binding. J. Mol. Biol. 384, 1232−1248. (15) Qian, M. X., Haser, R., and Payan, F. (1993) Structure and molecular-model refinement of pig pancreatic alpha-amylase at 2.1 Å resolution. J. Mol. Biol. 231, 785−799. (16) Janecek, S., Svensson, B., and MacGregor, E. A. (2003) Relation between domain evolution, specificity, and taxonomy of the alphaamylase family members containing a C-terminal starch-binding domain. Eur. J. Biochem. 270, 635−645. (17) Higuchi, R., Krummel, B., and Saiki, R. K. (1988) A general method of in vitro preparation and specific mutagenesis of dNA fragments - study of protein and DNA interactions. Nucleic Acids Res. 16, 7351−7367. (18) Rydberg, E. H., Sidhu, G., Vo, H. C., Hewitt, J., Cote, H. C., Wang, Y. L., Numao, S., MacGillivray, R. T. A., Overall, C. M., Brayer, G. D., and Withers, S. G. (1999) Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris. Protein Sci. 8, 635−643. (19) Bernfeld, P. (1955) Amylases, alpha and beta. Methods Enzymol. 1, 149−158. (20) Bozonnet, S., Jensen, M. T., Nielsen, M. M., Aghajari, N., Jensen, M. H., Kramhoft, B., Willemoes, M., Tranier, S., Haser, R., and Svensson, B. (2007) The ’pair of sugar tongs’ site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity. FEBS J. 274, 5055−5067. (21) Glaring, M. A., Baumann, M. J., Abou Hachem, M., Nakai, H., Nakai, N., Santelia, D., Sigurskjold, B. W., Zeeman, S. C., Blennow, A., and Svensson, B. (2011) Starch-binding domains in the CBM45 family - low-affinity domains from glucan, water dikinase and alpha-amylase involved in plastidial starch metabolism. FEBS J. 278, 1175−1185. (22) Numao, S., Damager, I., Li, C. M., Wrodnigg, T. M., Begum, A., Overall, C. M., Brayer, G. D., and Withers, S. G. (2004) In situ

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00992. Oligonucleotide sequences of the mutagenic primers (Table S1) and experimental procedure for protein mass spectrometric analysis and mass spectra of HPA and 4′O-methyl-α-maltosyl epi-cyclophellitol-HPA (MeG2ECP-HPA) (Figure S1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 604-822-3402. E-mail: [email protected]. Funding

This work was supported by an operating grant from the Canadian Institutes of Health Research [CIHR, Reference Number (FRN) 111082 (to G.D.B. and S.G.W.)]. S.G.W. is supported by a Tier One Canada Research Chair and the Canada Council Killam Fellowships program for salary support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Nham T. Nguyen for technical assistance. Portions of this research were conducted at the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, which is supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41GM103393).

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ABBREVIATIONS SBS, surface binding site; HPA, human pancreatic α-amylase; CNP-G3, 2-chloro-4-nitrophenyl α-maltotrioside; MbA, Montbretin A; MeG2-ECP, 4′-O-methylmaltosyl epicyclophellitol; G8, maltooctaose.

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