Báo cáo khoa học: Human salivary a-amylase Trp58 situated at subsite )2 is critical for enzyme activity potx

Human salivary a-amylase Trp58 situated at subsite )2 is critical for enzyme activity Narayanan Ramasubbu 1 , Chandran Ragunath 1 , Prasunkumar J. Mishra 1 , Leonard M. Thomas 2 , Gyo¨ ngyi Gye ´ ma ´ nt 3 and Lili Kandra 3 1 Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA; 2 Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, CA, USA; 3 Department 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 k cat ,anincrease in K m and 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, )3and+2 and showed an increase in transglycosylation activity com- pared with the wild type. The lowered affinity at subsites )2and)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) 8 barrel 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) Eur. J. Biochem. 271, 2517–2529 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04182.x Subsite 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 )2and)3 interact with residues Trp58, Trp59 and Gln63 (contained in a loop connecting b2anda2) 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 )3and )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 Trp58 fi Ala (W58A), Trp58 fi Leu (W58L) and Trp58 fi 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¢,whereXXX 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 DH10BAC TM 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 SETOR [48]. 2518 N. Ramasubbu et al. (Eur. J. Biochem. 271) Ó FEBS 2004 (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 Reagent TM (Invitrogen). After 72 h of incubation at 28 °C 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 28 °C 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 l M filter (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 m M Tris/ HCl, pH. 8.0. Following dialysis against a buffer (5 m M Tris/HCl, pH 8.0) containing 2 m M CaCl 2 , 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 25 °C for 3 min in 20 m M phosphate buffer (pH 6.9) containing 6 m M NaCl using 1% soluble starch as substrate [23]. Kinetic measurements were carried out using 4-nitrophenyl-a- D -maltoheptaoside (G 7 -PNP; Boehringer Mannheim) and p-nitrophenyl-a- D -maltopentaoside (G5- PNP; Sigma) in a coupled assay with 20 UÆmL )1 of 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– 5m M . The concentration of the wild type was 2 n M and concentration of the mutants W58A, W58Y and W58L was 20 n M 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 m M HEPES buffer (pH 7.1) containing 50 m M NaCl and 10 m M CaCl 2 at 30 °C. 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 m M ) 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 n M (HSAmy) or 500 n M (W58A, W58Y and W58L), oligosaccharide at 0.5 m M (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 m M each 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 m M glycerophosphate buffer (pH 7.0) containing 5m M Ca(OAc) 2 and 50 m M NaCl were carried out at 37 °C for 30, 40 and 60 min for W58L. The reactions were initiated by the addition of enzyme (final concentration of 1.85 n M HSA and 18.8 n M for the mutant W58L) to the solution containing 1.0 m M of 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 Ó FEBS 2004 Trp58 mutants at subsite )2 of human salivary a-amylase (Eur. J. Biochem. 271) 2519 flow rate of 1 mLÆmin )1 at 40 °C 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 )1 in 10 m M Tris/HCl (pH 9.0) containing 5 m M CaCl 2 was used. The reservoir solution contained 40% 2-methyl-2,4-pentanediol (MPD) and the hanging drops consisted of 2 lLofproteinand 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 m M final 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 K a radiation (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 a1° 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 R free 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 programs PROCHECK [32], CCP 4and CNS were 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. Parameters W58L W58A–acarbose complex Space group P2 1 2 1 2 1 P2 1 2 1 2 1 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 Multiplicity 3.8 6.0 R merge (%) (overall/last shell) a 6.2/28.1 6.9/23.1 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/R free (%) b 16.6/19.8 15.5/19.3 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. 2520 N. Ramasubbu et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Results Kinetics 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-PNP), the k cat values were lower significantly. Interestingly, although the K m values for the mutants were similar to the wild type for the substrate G7-PNP, the corresponding K m values for the substrate G5- PNP were higher. Thus, for the G5 substrate, there is an increase (5-fold) in the K m and a decrease in k cat (30–500- fold) compared with HSAmy. The k cat /K m valueforthetwo 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 k cat /K m for 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 typeforstarchaswellasG7-PNPassubstrates.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 m M , 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-andb-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 cat /K m 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 )1 Æm M )1 ) k cat (s )1 ) K m (m M ) k cat /K m (s )1 Æm 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]. Ó FEBS 2004 Trp58 mutants at subsite )2 of human salivary a-amylase (Eur. J. Biochem. 271) 2521 mutants, 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 )2and)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. 2522 N. Ramasubbu et al. (Eur. J. Biochem. 271) Ó FEBS 2004 more 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 )2and)1 at the nonreducing ends are occupied, occurs in the mutant W58L than in wild type. Also, the binding at the subsites )3and)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 conformation 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. Ó FEBS 2004 Trp58 mutants at subsite )2 of human salivary a-amylase (Eur. J. Biochem. 271) 2523 observed when compared with either W58L or HSAmy except for an altered orientation of the His305 side chain compared with HSAmy/acarbose complex (v 2 171°,W58A/ acarbose vs. v 2 )114°, HSAmy/acarbose complex). This altered conformation alone could not account for the significant reduction in the k cat as a mutation of His305 to Ala reduced the k cat by 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, )3and)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, )3and )2 subsites resulted in an increase in R free as 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 k cat and an increase in K m for 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 )2and )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, )3and)2 suggests that glucose units at these subsites might be highly positionally disordered. The positional disorder around subsites )2and)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 CNP-G 3 W58L 47 53 HSAmy – – CNP-G 4 W58L 13 74 13 HSAmy 10 85 5 CNP-G 5 W58L2612710 HSAmy 2 86 12 CNP-G 6 W58L2353028 5 HSAmy 51 44 5 CNP-G 7 W58L1133338132 HSAmy 18 50 30 2 2524 N. Ramasubbu et al. (Eur. J. Biochem. 271) Ó FEBS 2004 attacked 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, )3and)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 )2in 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 )2and)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. Ó FEBS 2004 Trp58 mutants at subsite )2 of human salivary a-amylase (Eur. J. Biochem. 271) 2525 pseudooligosaccharides. The residue Trp58 occurs in a loop region following the b2strandof(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. 2526 N. Ramasubbu et al. (Eur. J. Biochem. 271) Ó FEBS 2004 [...]... the subsite )2 glucose moiety and also assist in ordering the water structure around these subsites (Fig 6) Thus, only in the presence of Trp58, the mobile loop along with His305 repositions to anchor glucose at )2 (and possibly )3) susbites at the binding pocket In this regard, Trp58 might be involved in the pathway in which information flows between substrate and the catalytic site through His305 [13]... low activity when Met53 located at subsite )2 was replaced either with Tyr or Trp in barley a-amylase [42] Whether such an effect occurs in HSAmy is beyond the scope of this report Nonetheless, it should be pointed out that mutation of Asp300 to Asn in human pancreatic a-amylase, wherein the charge polarization could not occur, also leads to the reduction in kcat/Km values similar to that obtained for. .. (2000) Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques Biochemistry 39, 4778–4791 13 Ramasubbu, N., Ragunath, C & Mishra, P.J (2003) Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase J Mol Biol 325, 1061–1076 14 Davies, G.J., Wilson, K.S & Henrissat, B (1997) Nomenclature for sugar-binding... (2002) Barley alphaamylase Met53 situated at the high-affinity subsite- 2 belongs to a substrate binding motif in the beta fi alpha loop 2 of the catalytic (beta/alpha) 8-barrel and is critical for activity and substrate specificity Eur J Biochem 269, 5377–5390 Ishikawa, K., Matsui, I., Honda, K & Nakatani, H (1992) Multifunctional roles of a histidine residue in human pancreatic alphaamylase Biochem Biophys... recombinant human salivary amylase Protein Exp Purif 24, 202–211 20 Mishra, P.J., Ragunath, C & Ramasubbu, N (2002) The mechanism of salivary amylase hydrolysis: role of residues at subsite S2¢ Biochem Biophys Res Commun 292, 468–473 21 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 Ó FEBS 2004 Trp58 mutants at subsite )2 of human salivary. .. alpha/beta barrel enzymes Trends Biochem Sci 15, 228–234 8 Brayer, G.D., Luo, Y & Withers, S.G (1995) The structure of human pancreatic alpha-amylase at 1.8 Angstrom resolution and comparisons with related enzymes Protein Sci 4, 1730–1742 9 Ramasubbu, N., Paloth, V., Luo, Y., Brayer, G.D & Levine, M.J (1996) Structure of human salivary a-amylase at 1.6 Angstrom resolution: implications for its role in... catalytic carboxyl group Asp300 suggests that some distortion of the negative electrostatic potential might be occurring at the catalytic site To test this, atomic charges on the carboxyl group atoms of Asp300 were evaluated by semiempirical methods (MOPAC with AM1 as provided in the SYBYL software, Tripos Inc.) The atomic charges on the atoms around position 58 in HSAmy, W58L and W58A were calculated... 2004 Trp58 mutants at subsite )2 of human salivary a-amylase (Eur J Biochem 271) 2527 Fig 6 Stereodiagram depicting the changes in the interactions around position 58 in W58A–acarbose complex (thin lines) as compared with the wildtype enzyme complex (thick lines) The mutation has resulted in minor changes in the side conformation of residues Lys352 and His305 Clear density for the side chain of His305... other a-amylases containing Trp58 and the mobile loop, Trp58 is required for wild-type kinetic properties especially in the affinity and in the hydrolysis of maltooligosaccharides Replacement of this residue with residues containing smaller side chain resulted in the decrease of enzyme activity Such a replacement also misguided the substrate binding in the glycone subsites )2, )3 and )4 to an extent that... compared (data not shown) This preliminary analysis suggested that the overall negative charge on the two oxygen atoms of the carboxylate group was higher in the wild type than in the mutants The positive charge on the carbon atom of the carboxylate group remained the same in all three structures Such distortion of negative electrostatic potential has been suggested to be partially responsible for the . Human salivary a-amylase Trp58 situated at subsite )2 is critical for enzyme activity Narayanan Ramasubbu 1 , Chandran Ragunath 1 , Prasunkumar J. Mishra 1 ,. at subsite )2in HSAmy) was required for wild-type kinetic properties such as affinity [42]. Inadequate binding at subsite )2 caused distortions at the subsite