Energetics of Substrate Binding and Catalysis by Class 1(Glycosylhydrolase Family 47) �-Mannosidases Involved in N-GlycanProcessing and Endoplasmic Reticulum Quality Control*□S
Received for publication, May 10, 2005, and in revised form, May 23, 2005Published, JBC Papers in Press, May 23, 2005, DOI 10.1074/jbc.M505130200
Khanita Karaveg and Kelley W. Moremen‡
From the Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology,University of Georgia, Athens, Georgia 30602
Nascent glycoproteins are subject to quality controlin the lumen of the endoplasmic reticulum (ER) wherethey can either be effectively folded with the aid of acollection of ER chaperones or they can be targeted fordisposal in a process known as ER-associated degrada-tion. Initiation of the ER disposal process involvesselective trimming of N-glycans by ER �-mannosidase Iand subsequent recognition by the ER degradation-enhancing �-mannosidase-like protein family of lec-tins, both members of glycosylhydrolase family 47. Thekinetics and energetics of substrate binding and catal-ysis by members of this family were investigated hereby the analysis of wild type and mutant forms of hu-man ER �-mannosidase I. The contributions of severalamino acid residues and an enzyme-associated Ca2�
ion to substrate binding and catalysis were demon-strated by a combination of surface plasmon reso-nance and enzyme kinetic analyses. One mutant,E330Q, shown previously to alter general acid functionwithin the catalytic site, resulted in an enzyme thatpossessed increased glycan binding affinity but com-promised glycan hydrolysis. This mutant protein wasused in a series of glycan binding studies with a libraryof mannose-containing ligands to examine the energet-ics of Man9GlcNAc2 substrate interactions. These stud-ies provide a framework for understanding the natureof the unusual substrate interactions within the family47 mannosidases involved in glycan maturation andER-associated glycoprotein degradation.
As polypeptide chains are extruded through the endoplasmicreticulum (ER)1 membrane during co-translational transloca-tion, they are commonly glycosylated on the amide side chains ofAsn residues within the acceptor consensus sequon, Asn-X-(Ser/Thr) (1). Trimming of terminal glucose residues results in the
formation of glycan structures that can act as ligands for theluminal ER lectin chaperones, calnexin and calreticulin (2, 3),which can aid in the folding of the nascent polypeptides in thelumen of the ER (2, 4). Glycoproteins that have slow foldingkinetics continually re-engage the lectin chaperones either untilfolding is complete (2, 4, 5) or until the nascent glycoproteinsacquire a target signal for disposal (6–8). For terminally mis-folded glycoproteins, trimming of the oligosaccharide by the ac-tion of ER �-mannosidase I (ERManI) to generate a uniqueMan8GlcNAc2 isomer product (Fig. 1E) is the key rate-limitinginitiation signal (9, 10) that ultimately leads to retrotranslocationof the polypeptide back into the cytoplasm for degradation by theproteasome in a process known as ER-associated degradation(ERAD) (11). Inhibition of ERManI can cause the accumulationof misfolded model glycoproteins in the ER lumen (12–20), andERManI overexpression has been shown to accelerate the “dis-posal clock,” hastening the disposal of misfolded proteins andeven early folding intermediates of wild type proteins (9). Thus,the efficiency of creating fully folded glycoproteins for transportfrom the ER is defined by a competition between the kinetics ofconformational maturation versus the rate of acquiring the keyglycan signal for glycoprotein disposal.
Many loss-of-function human genetic diseases result fromdelayed folding kinetics of potentially functional polypep-tides, such as the �Phe508 mutant of cystic fibrosis trans-membrane regulator (21), rather than generating terminallymisfolded protein structures (22). Thus, treatment of manyprotein misfolding disorders could be achieved if pharmaco-logical inhibition of the rate-determining steps for ERADallowed sufficient time for completion of the protein foldingprocess (7, 23).
ERManI is a member of a larger family of proteins, termedClass 1 mannosidases (24) (CAZy family 47 glycosylhydrolases(25–28)), involved in glycoprotein maturation and disposal.Two other subgroups within this family include a subfamily ofhydrolases in the Golgi complex and a subfamily of lectins inthe ER. The Golgi �-1,2-mannosidases (termed IA (29), IB (30),and IC (31)) are essential for trimming high mannose N-gly-cans to the Man5GlcNAc2-Asn intermediate necessary for mat-uration into complex type structures on cell surface and se-creted glycoproteins. In the ER, the EDEM subgroups ofproteins apparently have no hydrolase activity but act as lec-tins as a part of the ERAD disposal machinery (2, 32–38). Thepresent models envisage recognition of the glycan structures bythe EDEM proteins in a mode similar to substrate recognitionduring catalysis by the true hydrolases, followed by transfer tothe Sec61 translocon pore, retrotranslocation into the cytosol,and proteasomal degradation (2). Thus, understanding howthis family of enzymes and lectins accomplish their functions inrecognition and catalysis will provide insights into the rate-
* This work was supported by National Institutes of Health ResearchGrants GM47533 and RR05351 (to K. W. M.). The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
□S The on-line version of this article (available at http://www.jbc.org)contains Tables 1 and 2 and Figs. 1–4.
‡ To whom correspondence should be addressed: Complex Carbohy-drate Research Center, University of Georgia, Athens, GA 30602. Tel.:706-542-1705; Fax: 706-542-1759; E-mail: [email protected].
1 The abbreviations used are: ER, endoplasmic reticulum; ERAD,endoplasmic reticulum-associated degradation; EDEM, ER degradationenhancing �-mannosidase-like protein; ERManI, ER �-mannosidase I;Golgi ManIA, Golgi �-mannosidase IA; dMNJ, 1-deoxymannojirimycin;Kif, kifunensine; HPLC, high performance liquid chromatography; PA,pyridylamine; SPR, surface plasmon resonance; ITC, isothermal titra-tion calorimetry; MES, 4-morpholineethanesulfonic acid.
FIG. 1. Model for the structure and catalytic residues of human ERManI used in the mutagenesis studies described in this paper.The end (A) and side (B) views of the human ERManI ribbon diagram (Protein Data Bank 1X9D (46)) display the (��)7 barrel structure with theN-glycan substrate (Man5GlcNAc2 substrate, stick representation from Protein Data Bank 1DL2 (43) and the glycone residue in the �1 subsite
Glycan Binding and Catalysis by �-1,2-Mannosidases29838
limiting decisions between glycoprotein maturation and dis-posal in the secretory pathway.
Class 1 �-mannosidases have been studied extensively withregard to enzyme kinetics, substrate specificity, structure, andmechanism (24, 39–46). The catalytic domains of enzymesfrom fungal and mammalian sources have similar (��)7 barrelstructures (41–45) that are plugged at one end by a �-hairpin,whereas the opposite end is composed of a broad cleft leading tothe catalytic residues in the barrel core (Fig. 1, A and B). In allof the enzymes, a Ca2� ion is bound at the apex of the �-hairpinin the core of the barrel where it is involved in direct interac-tions with two of the glycone hydroxyls during the catalyticcycle (Fig. 1, A–D) (42, 46). Co-complex structures have beenexamined between ERManI and the glycone mimic inhibitors,1-deoxymannojirimycin (dMNJ) and kifunensine (Kif) (Fig. 1F)(42), as well as a co-complex with an uncleaved thiodisaccha-ride pseudosubstrate (46), revealing a novel conformationalitinerary during glycoside bond hydrolysis. Our recent studiessuggest that the enzyme binds to the glycone residue in the �1subsite in a high free energy 3S1 conformation (46), whichallows the formation of a ring-flattened 3H4 transition state bya least motion conformational twist of the predisposed sugarring, and produces an inverted enzymatic product in a 1C4
conformation. Novel general base (Glu599) and general acid(Glu330 and Arg334 acting in a through-water protonationscheme) functions were identified through a combination ofkinetic analyses and structure determination of the ERManI-thiodisaccharide co-complex (Fig. 1, C and D) (46). All of theknown Class 1 mannosidase structures are essentially identi-cal within the �1 and �1 subsites, suggesting that the catalyticmechanism for bond hydrolysis is conserved among all of thetrue hydrolases.
Although all of the Class 1 mannosidases cleave Man-�1,2-Man linkages, there are significant differences in branch spec-ificities among the different family members (24). ERManIcleaves a single residue from the central branch of theMan9GlcNAc2 substrate to produce a single Man8GlcNAc2 iso-mer product (Fig. 1E) (39, 40, 47). In contrast, the Golgi sub-family of enzymes recognizes the other terminal Man-�1,2-Manbranches but instead cleaves the central branch with poorefficiency (48, 49). Thus, these enzymes have a mutually exclu-sive but complementary specificity for the complete cleavage ofMan-�1,2-Man linkages on high mannose glycans (41). Puta-tive glycan enzymatic product co-complexes have been isolatedfor members of both the ER (43) and Golgi (41) subclasses ofenzymes, demonstrating that differences in the cleft structuresleading from the catalytic core residues confer unique glycanbranch specificities for the different subfamily members.
In the studies described here, we have complemented andextended our recent work on the characterization of the ER-ManI catalytic mechanism (46) by examining for the first timethe energetics of substrate binding and catalysis by a class 1
�-mannosidase. Kinetic and binding analyses have allowed usto dissect the energetic contributions of individual amino acidresidues and the protein-bound Ca2� ion during substratebinding and catalysis. Through the use of a general acid cata-lytic mutant that is compromised in hydrolysis, yet maintainssubstrate binding with similar characteristics to the wild typeenzyme, we also mapped the contributions of individual resi-dues in the Man9GlcNAc2 substrate for their interactions withthe extended enzyme glycan binding pocket. These studiesrevealed unanticipated roles for glycan interactions in the ��1 subsites for facilitating catalysis and substrate specificity.The experimental strategy for the binding and kinetic analysesalso provides a framework for further studies on the structuraland energetic basis of substrate branch recognition and catal-ysis by other members of the class 1 (glycosylhydrolase family47) �-mannosidases.
MATERIALS AND METHODS
Mutagenesis, Expression, and Purification of Human ERManI—Themutagenesis, expression, and purification of the human ERManI cata-lytic domain has been described previously (42, 46). Briefly, the cDNAencoding the human ERManI catalytic domain in the pPICZ�A vector(Invitrogen) was used to perform site-directed mutagenesis using theQuikChangeTM mutagenesis kit from Stratagene (La Jolla, CA). Plas-mid constructs were then used to transform the Pichia pastoris strainX-33, and Zeocin-resistant colonies were screened for ERManI expres-sion by performing Western blots using conditioned medium from in-duced cultures as described previously (42, 46). Mutant enzymes wereexpressed in 1-liter shake flask cultures by induction in BMMY media,and the enzyme was purified from the conditioned media as describedpreviously (42, 46).
Glycopeptide and Glycan Isolation—Man9GlcNAc2-glycopeptideswere isolated from crude soybean agglutinin by reduction, carboxy-amidomethylation, elastase digestion, affinity chromatography usingconcanavalin A-Sepharose, and further purification by HPLC on aCosmosil C18 column as described previously (46). Man9GlcNAc2 wasliberated from the peptide by peptide:N-glycosidase F digestion (50)and derivatized with pyridylamine (Man9GlcNAc2-PA) (48).Man8GlcNAc2-PA isomers, Man6GlcNAc2-PA, and Man5GlcNAc2-PA,were generated by digestion with either ERManI or Golgi ManIA andisolation by reverse phase HPLC as described (48).
Enzyme, Protein, and Carbohydrate Assays—The purified wild typeand mutant enzymes were assayed for �1,2-mannosidase activity usingMan9GlcNAc2-PA as substrate as described previously (46). Briefly, theenzyme reactions (20 �l) were carried out in 96-well plates at 37 °C forthe indicated times, stopped by the addition of 20 �l of 1.25 M Tris-HCl,pH 7.6, and resolved and quantitated using a Hypersil APS-2 NH2-HPLC column (48). One unit of enzyme activity is defined as theamount of enzyme that generates 1 �mol of Man8GlcNAc2 fromMan9GlcNAc2 in 1 min at 37 °C. Protein concentration was determinedusing the BCA protein assay reagent (Pierce) as described by themanufacturer. Oligosaccharide concentrations were determined by phe-nol-sulfuric acid assays (51).
Kinetic Analysis—Initial rates (v) for the enzymes were determinedat various substrate concentrations ranging from 10 to 300 �M. Thecatalytic coefficient (kcat) and Michaelis constant (Km) values weredetermined by fitting initial rates to a Michaelis-Menten function bynonlinear regression analysis using SigmaPlot (Jandel Scientific, San
from Protein Data Bank 1X9D (46)) bound in the core of the barrel highlighted by the dark blue circle. The glycone residue in the �1 subsite (lightblue circle, B) is in direct association with the protein-bound Ca2� ion (blue spacefill). The residues examined in this study are shown in the stereodiagram (C) where the stick representation of the Man6GlcNAc2 glycan substrate is shown in yellow, the Ca2� ion is shown as a blue spacefill, andthe relevant residues described in the text are shown as stick diagrams. Residues mutated in this study are shown as green stick figures, whereasthe Glu330 residue studied previously (46) is shown as a light blue stick figure. Water molecules coordinating the Ca2� ion are indicated by smallred spacefill structures with interactions with the Ca2� ion indicated by cyan dotted lines. Interactions between the carbonyl oxygen and O-� ofThr688 and the Ca2� ion are also represented by cyan dotted lines. Proposed acid, base, and nucleophile trajectories as described previously (46)are illustrated with magenta dotted lines. Hydrogen bonds are shown as green dotted lines. A schematic diagram demonstrating the interactionsbetween the N-glycan and the active site (D) employ a similar color scheme for hydrogen bonding, acid, base, and nucleophile trajectories and Ca2�
coordination as C, with the exception of the hydrophobic stacking between Phe659 and the C4-C5-C6 region of the �1 residue (black dotted lines).Residue numbering of amino acid side chains in the respective subsites is indicated in the figure. The residue nomenclature and linkages for themonosaccharides in the Man9GlcNAc2-Asn substrate are indicated (E), and the linkage cleaved by ERManI is also indicated. A similar monosac-charide nomenclature is used in C and D to label the respective residues in the glycan structure. Labeling of the enzyme subsites with negative(glycone in the �1 subsite) or positive (�1 and �2 subsite residues) numbers reflects their respective positions relative to the glycosidic bond beingcleaved (64). Schematic structures of �-D-mannose and the corresponding inhibitors, dMNJ and Kif, are shown in F.
Glycan Binding and Catalysis by �-1,2-Mannosidases 29839
Rafael, CA). kcat/Km values were derived from reciprocal plots of v and[S] where needed. In studies on the temperature dependence of catal-ysis, values for kcat were obtained between 5 and 40 °C at 5 °C intervalsand were used to calculate activation energies (Ea) from the slopes(�Ea/R) of Arrhenius plots (ln(kcat) as a function of 1/T). The thermo-dynamic activation parameters were described by the Equations 1–3(52, 53):
�G‡ � RT � � lnkBTh
� lnkcat� (Eq. 1)
�H‡ � Ea � RT (Eq. 2)
�S‡ � ��H‡ � �G‡�/T (Eq. 3)
where R is the gas constant (8.314 J�mol�1 K�1); kB is the Boltzmannconstant (1.3805 �10�23 J�K�1), and h is the Planck constant (6.6256 �10�34 J�s).
Calcium Equilibrium Analysis—Purified wild type ERManI or theT688A mutant was incubated with 200 mM EGTA for 2 h at 4 °C priorto desalting over a Sepharose G-25 column (1 � 40 cm), which waspretreated with 0.5 M EGTA, followed by pre-equilibration in calcium-free buffer (20 mM MES, pH 7.0, 150 mM NaCl). The calcium-freeprotein solution was concentrated to 1 mg/ml using an Amicon YM-10membrane. The calcium content of the protein solution, confirmed byinductively coupled plasma mass spectrometry (ICP-MS), was below50 ppb.
The total amount of calcium chloride required to generate definedconcentrations of free Ca2� ion (0–500 �M) in an EGTA-containingbuffer (20 mM MES, pH 7.0, 150 mM NaCl, 5 mM EGTA) was calculatedusing WEBMAXCLITE version 1.15 (54) (available online at www.stanford.edu/�cpatton/maxc.html). Solutions of defined Ca2� ion con-centration were prepared, and the pH of the mixture was monitored andadjusted to pH 7.0 by addition of NaOH. All stock solutions wereprepared in EDTA-treated plasticware. Total calcium ion concentra-tions in each solution were confirmed by atomic absorption and used torecalculate the free calcium ion concentration under the conditions ofanalysis (55).
The calcium-free enzyme solution was diluted in calcium-free bufferto obtain a stock solution of 80 �g/ml for wild type ERManI and 140�g/ml for the T688A mutant. Aliquots of the enzyme solutions (5 �l)were added to each of the buffers (10 �l) containing defined Ca2� ionconcentrations before addition of 5 �l of 80 �M Man9GlcNAc2-PA. En-zyme reactions were allowed to proceed at 37 °C for 1 h, stopped, andanalyzed by NH2-HPLC chromatography as described above. Plotting ofmannosidase enzyme activity versus Ca2� ion concentration revealed asigmoidal curve similar to data expected for a common equilibriumdialysis experiment, allowing the calculation of the Ca2� affinity con-stant (KCa) by nonlinear regression analysis using Equation 4 (55),
y �nKCa�Ca2�
1 KCa�Ca2�(Eq. 4)
where y is equal to the moles of Ca2� bound per mol of enzyme,measured as units of �-mannosidase enzyme activity resulting fromCa2� binding to the enzyme, and n is the apparent ERManI-specificactivity.
Binding Studies by Surface Plasmon Resonance (SPR)—SPR analy-ses were conducted using a Biacore 3000 apparatus (Biacore AB) withrecombinant ERManI immobilized on the SPR chip surfaces at 25 °C bythe amine-coupling method as described previously (46). Mock-derivat-ized flow cells served as reference surfaces. The binding analyses wereroutinely performed at 10 °C with continuous flow (30 �l/min) of run-ning buffer except in the temperature-dependent interaction studies,which were performed between 5 and 35 °C at 5 °C intervals, consecu-tively, in an automated method. The running buffer was 10 mM MES,pH 7.0, 300 mM NaCl, and 5 mM CaCl2 in all cases, except in calcium-dependent binding studies. For the latter studies, the chip surface wassubjected to overnight treatment with 20 mM MES/NaOH, pH 7.0, 300mM NaCl, 5 mM EGTA, at a flow rate of 5 �l/min, prior to bindinganalyses using 20 mM MES/NaOH, pH 7.0, 300 mM NaCl, and 200 �M
EGTA as running buffer. Analytes were prepared in the respectiverunning buffers by 2-fold serial dilution to obtain an appropriateconcentration range. The binding of Man9–5GlcNAc2-PA andMan9GlcNAc2-glycopeptide glycans was analyzed in a concentrationseries (0.4–400 �M) over a low density immobilization surface (46) ofrecombinant protein (3000 response units), whereas dMNJ and Kif(2–1000 �M) were analyzed over a high density immobilization surface
(46) of recombinant protein (10,000 response units). The base linereturned to the original response in 5 min for all analytes describedhere without a further regeneration procedure, except for analysesusing Kif as the analyte, which did not dissociate from the chip surfaceeven with extensive washing.
SPR data for each concentration of analyte were collected in dupli-cate and globally fit to a 1:1 Langmuir binding algorithm model tocalculate the on-rate (ka), the off-rate (kd), and the equilibrium dissoci-ation constant (kd/ka KD) using the BIAevaluation 3.1 software (56).Alternatively, the maximal equilibrium sensorgram values were used toplot a saturation binding curve and calculate values for the equilibriumdissociation constant (KD) directly.
KD values measured at different temperatures were used to calculatethermodynamic parameters (56, 57) of binding using van’t Hoff equa-tion (Equation 5), (58) and Gibbs free energy change for binding (�G)was also calculated (Equation 6). The van’t Hoff equation allows thecalculation of thermodynamic parameters using the linear relationshipof y ln(KD) versus x 1/T, which gives a slope of �H/R and anintercept of ��S/R (58).
lnKD � �H/�RT� � �S/R (Eq. 5)
�G � �RT ln �1/KD� (Eq. 6)
The effects of temperature on the association rates (ka) and dissoci-ation rates (kd) were independently determined using the Eyring equa-tion (Equation 7) (58).
k � �kBT/h�exp��S†/R�exp� ��H †/RT� (Eq. 7)
Similar to the van’t Hoff analysis, the Eyring equation allows thermo-dynamic parameters to be determined from measured ka and kd valuesat different temperatures by a linear relationship of y Rln(hka/kBT) ory Rln(hkd/kBT) versus x 1/T, where the slope and the intercept ofthe Eyring plots are ��H† and �S†, respectively (58).
Isothermal Titration Calorimetry (ITC)—Calorimetry measurementswere performed with a 4200-ITC calorimeter (Calorimetry SciencesCorp., Lindon, UT) as described (59, 60). Protein solutions for ITCanalysis were dialyzed overnight against buffer containing 20 mM MES,pH 7.0, 150 mM NaCl, 5 mM CaCl2 and 0.75 M 3-(1-pyridino)-1-propanesulfonate (NDSB201; Calbiochem) at 4 °C. The ligand solutions of Kifand dMNJ were prepared by diluting the compounds in the buffer usedfor protein dialysis. Aliquots (5–10 �l) of the ligand solution (1–5 mM)were automatically delivered into 1.3 ml of protein solution (1–2 mg/ml)in the reaction cell. The calorimetry cell was allowed to return toequilibrium for 4 min prior to the next injection. The data analysis wasperformed using DataWork and BindWork software provided by man-ufacturer. Protein and glycan molecular structure figures were pre-pared using MacPymol (version 0.95)2 to generate rasterized images.
RESULTS
Kinetic Analysis of ERManI Mutants—Mutations were gen-erated previously in five residues that were hypothesized to beinvolved in catalysis by ERManI (46). These mutations testedthe roles of putative general acid (E330Q and R334A) andgeneral base (E599Q and H524A) functions, as well as a resi-due that proved to play a critical role in providing hydrogenbonding interactions with the mannose residue in the �1 sub-site (D463N) (Fig. 1, C and D). In the present study, we havemutagenized two residues at the base of the �1 subsite (Phe659
and Thr688), one residue involved in interactions with several ��1 residues (Arg461), and one residue that interacts with boththe �1 and �2 subsite residues (Arg597) (Fig. 1, C and D), andcharacterized their roles in catalysis and substrate binding.Phe659 was shown previously to provide van der Waals inter-actions to the C4-C5-C6 region of the glycone in the �1 subsiteduring catalysis (Fig. 1D) (42, 46). Thr688 is positioned at theapex of the �-hairpin in the core of the (��)7 barrel where it hasbeen shown to be the sole protein residue that directly coordi-nates the bound Ca2� ion through both its O-� and carbonyloxygens (Fig. 1D) (42, 46). The other four points of coordinationof the Ca2� ion to the enzyme are indirect through-water in-
2 W. L. Delano, Pymol Molecular Graphics System, available onlineat www.pymol.org.
Glycan Binding and Catalysis by �-1,2-Mannosidases29840
teractions with carboxylate side chains in the core of the barrel.Arg461 interacts with several residues in the core of theMan9GlcNAc2 substrate (Fig. 1, D and E, residues M7, M4, andM3) and has been proposed to contribute to branch specificityfor Saccharomyces cerevisiae ERManI (61). A Leu residue isfound at the equivalent position in the Golgi subclass of en-zymes. Previous mutagenesis studies generating the equiva-lent of an R461L mutant for S. cerevisiae ERManI (61) resultedin an enzyme that had a hybrid activity between the specificityof ERManI that cleaves only the central branch mannose res-idue and the Golgi mannosidases that cleave the remaining�1,2-Man residues down to Man5GlcNAc2. Finally, Arg597 ap-pears to play a dual role in hydrogen bonding to the O-6�hydroxyl oxygen of the glycone in the �1 subsite via NH1 andthe O-4� hydroxyl of the mannose in the �2 subsite via NH2.Each of the mutants (T688A, F659A, R461A, R461L, andR597A) was expressed in P. pastoris as a secreted catalyticdomain, and the detailed enzyme kinetic parameters for hy-drolysis of Man9GlcNAc2-PA were determined and are sum-marized in Table I. The pH optima for all of the mutantenzymes were slightly below the value for wild type ERManI(pH 6.5–6.8 versus 7.0 for wild type; Table I). The catalyticrates (kcat) and the catalytic efficiencies (kcat/Km) ofMan9GlcNAc2 cleavage for all of the mutants were signifi-cantly decreased, resulting in a range of kcat/Km values thatvaried from 0.6 to 12% of wild type values. Surprisingly, theKm value for the T688A mutant was reduced 7.3-fold,whereas the Phe659 mutant remained unaffected. In contrast,the Km values for the other mutants were all significantlyincreased by 3–5-fold.
For all of the mutants tested, the R461L mutant wasunique in its ability to readily hydrolyze �1,2-mannosideresidues from Man9GlcNAc2-PA to Man8–6GlcNAc2-PA, asdescribed previously for an equivalent mutant of S. cerevisiaeERManI (61) (data not shown). The 53-fold decrease in kcat
and 3-fold increase in Km (Table I) indicated that althoughthe amino acid substitution relaxed the specificity of theenzyme for glycan cleavage beyond Man9GlcNAc2, the en-zyme lost significant catalytic efficiency as a result of themutation. In contrast, the R461A or R597A mutants wereunable to cleave beyond Man8GlcNAc2. However, the cata-lytic rate of the R461A mutant was intermediate between thewild type enzyme and the R461L mutant. These data suggestthat the removal of the Arg461 side chain in the R461Amutant moderately compromised catalysis while retainingERManI substrate specificity. In contrast the R461L mutantwas altered in substrate specificity, although its catalyticefficiency was severely compromised.
The catalytic rates (kcat) of wild type ERManI and the E330Qand T688A mutants were also obtained from initial rates meas-ured at different temperatures but under optimal pH condi-tions for the respective enzymes (pH 7.1, 5.3, and 6.5 for wild
type, E330Q, and T688A, respectively). The enzyme activitiesincreased with temperature, and activation energies (Ea)were calculated from the slopes of the Arrhenius plots (sup-plemental Fig. 1A and supplemental Table I). The wild typeenzyme and T688A mutant appear to have similar trends,with significant enthalpy and entropy contributions to theactivation energy, whereas the E330Q mutant had a slightlyreduced entropic contribution (supplemental Fig. 1A and sup-plemental Table I).
Effect of the T688A Mutant of ERManI on Ca2� Ion Affinityand Enzyme Activity—The role of the protein-bound Ca2� ionin catalysis by ERManI was determined by depleting wild typeERManI or the T688A mutant of bound Ca2� and then per-forming enzyme assays at defined Ca2� concentrations con-trolled by the presence of the divalent cation chelator EGTA. Inthe absence of any added Ca2�, both proteins exhibited nodetectable enzyme activity (supplemental Fig. 2). Addition ofCa2� resulted in a progressive appearance of enzyme activity,allowing the calculation of the Ca2� affinity for the enzyme,KCa, by curve-fitting. The KCa values for the wild type enzyme(0.24 � 0.02 �M) and the T688A mutant (0.15 � 0.01 �M) werequite similar, yet the specific activity of the T688A mutantwas generally �15-fold lower than the wild type enzyme atall Ca2� concentrations where activity could be detected. Theobservation that the T688A mutant is compromised in catal-ysis (reduced kcat) but increased in substrate binding affinity(reduced Km), while being essentially unaltered in Ca2� bind-ing affinity, indicates that the mutation has a direct effect oncatalysis rather than acting through a reduced affinity forbinding and coordinating Ca2�. The KCa value for wild typeERManI was similar to the affinity constants previously de-termined for yeast ERManI (62) and rabbit liver Golgi ManIA(63).
Glycan and Inhibitor Binding Affinity Measurements to Hu-man ERManI—In addition to kinetic analysis, the bindingaffinities of inhibitors and high mannose oligosaccharides towild type and mutant forms of ERManI were also examined bySPR. Prior SPR studies with wild type and mutant forms ofERManI revealed significant alterations in the on-rates (ka)and off-rates (kd) of binding to dMNJ and Man9GlcNAc2-glyco-peptide ligands (46). Correlations were made with the positionsof the mutations and their impacts on ligand binding affinity.In the present study, we performed similar types of SPR stud-ies with wild type ERManI in the presence and absence ofCa2�, as well as testing the effects of the T688A, F659A,R597A, R461L, and R461A mutants on the binding ofMan9GlcNAc2-glycopeptide, dMNJ, or Kif ligands (Fig. 2 andTable II). As described previously (46), the equilibrium disso-ciation constants (KD) could be measured from a combination ofthe on-rates (ka) and off-rates (kd) determined by curve-fittingof the SPR sensorgrams, where KD kd/ka. When the on-ratesand off-rates were too fast for accurate measurement, plotting
TABLE IKinetic constants for wild type and mutant human ERManI using Man9GlcNAc2 as substrate
a Assay data were fit to generate a bell-shaped curve to yield a pH optimum with a standard error of 0.1 pH unit.b Kinetic constants for wild type and E330Q mutant of ERManI were as reported previously (46) and are shown as a reference.c The enzyme exhibits the ability to readily hydrolyze Man9GlcNAc2-PA to Man6GlcNAc2-PA.
Glycan Binding and Catalysis by �-1,2-Mannosidases 29841
of a saturation curve for the equilibrium values of the bindingsensorgrams (Fig. 2, inset plots) allowed an alternative meansof determining the KD values (46).
Depletion of Ca2� from wild type ERManI resulted in asignificantly slowed on-rate and off-rate for binding of theMan9GlcNAc2-glycopeptide ligand by SPR (Fig. 2), but resultedin only a 1.6-fold reduction in the equilibrium binding affinityfor the glycan ligand (Table II). In contrast, the binding affinityof dMNJ was reduced 343-fold, largely as a result of a 127-foldreduction in the on-rate (Table II). These data indicate that thepresence of Ca2� bound to the core of the (��)7 barrel influencesthe rate of glycone binding to the �1 subsite, but the overallequilibrium binding affinity of the larger Man9GlcNAc2 sub-strate is not significantly influenced by the absence of thedivalent cation.
Similar to the reduced on- and off-rates for Man9GlcNAc2-glycopeptide binding to the Ca2�-depleted enzyme, the T688A
mutant also had significantly reduced on- and off-rates for highmannose glycan binding, but an �50-fold increase in the equi-librium binding affinity (Table II) similar to the increase inglycan binding affinity previously observed for a E330Q gen-eral acid mutant (46). The increased glycan binding affinity forthe T688A mutant, when combined with the significantly re-duced kcat (Table I) and an unaltered Ca2� binding affinity(supplemental Fig. 2), indicates that the reduced catalytic turn-over results in a substrate that is stabilized in an uncleavedform in the active site. This altered environment is less favor-able for binding to dMNJ (5-fold decrease in binding affinity),which we have proposed previously (42, 46) to resemble theconformation of the enzymatic product.
In contrast to the effects of the T688A mutant at the base ofthe �1 subsite, mutation of Phe659 (F659A), which providesvan der Waals interactions with the �1 subsite residue, had aminimal effect on Man9GlcNAc2 binding (4-fold reduction in
FIG. 2. SPR binding of Man9Glc-NAc2, dMNJ, or Kif ligands to wildtype (WT) and mutant ERManI. Wildtype or mutant ERManI forms wereimmobilized on the SPR chip surfaceas described under “Materials andMethods,” and various concentrations ofMan9GlcNAc2 (A, C, E, and G), dMNJ (B,D, F, and H) or Kif (I and J) ligands weretested for binding. The data were col-lected in duplicate, and representativeSPR sensorgrams in the ligand concen-tration series are shown. If the on- andoff-rates (ka and kd, respectively) weresufficiently slow, curve fitting of the sen-sorgrams was performed using the 1:1Langmuir binding algorithm model todetermine the values for the equilibriumdissociation constants (KD kd/ka). Inbinding studies where the kineticsfor binding of the Man9GlcNAc2 ligandwere too rapid for curve fitting, the equi-librium sensorgram values were used toplot a saturation curve (insets in the Aand G plots) and calculate values for KD.In the F659A mutant, binding of dMNJwas completely abolished as indicated bythe absence of a deflection in the SPRsensorgram trace. The values for ka, kd,and KD based on the SPR studies areshown in Table II.
Glycan Binding and Catalysis by �-1,2-Mannosidases29842
KD, see Table II), consistent with the lack of an effect of themutation on the Km with the same glycan substrate. However,the binding of dMNJ was completely abolished for this mutantenzyme (KD � 10 mM; Fig. 2), consistent with 123-fold reduc-tion in kcat (Table I). These data suggest that Phe659 plays a keyrole in glycone binding within the �1 subsite to promotecatalysis.
Finally, the binding of Kif to wild type ERManI was exam-ined by SPR. The equilibrium binding affinity for this inhibitorcould only be estimated at 30 nM, because there was nodetectable dissociation of the compound from the enzyme, evenafter extensive washing (Fig. 2). Inhibitor binding studies byITC confirmed the tight binding by the inhibitor, which washighly exothermic and had a strongly favorable enthalpy ofbinding (see supplemental Table II). The E330Q, D463N, andT688A mutants also formed stable nondissociable complexeswith Kif (data not shown). To test the role of the van der Waalsinteractions between Phe659 and the inhibitor at the base of the�1 subsite, we examined the binding of Kif to the F659Amutant. In contrast to the wild type enzyme, binding of Kif tothe F659A mutant was reversible (Fig. 2), with measurable on-and off-rates and a KD of 1.45 �M (Table II).
Binding of Man9GlcNAc2 to the R461A and R597A mutantsexhibited reduced on- and off-rates, but the KD values weresimilar to the wild type enzyme (data not shown). In contrast,the R461L mutant exhibited no detectable binding (KD � 1mM) to Man9GlcNAc2 (data not shown), consistent with thepoor catalytic efficiency of this mutant enzyme that has ahybrid activity between ERManI and the Golgi subclass ofenzymes.
Temperature and pH Dependence of Glycan Binding—In aneffort to examine the temperature dependence of glycan bind-ing to the wild type and mutant forms of ERManI, we per-formed a series of SPR binding studies with Man9GlcNAc2 asligand at temperatures between 5 and 35 °C. The sensorgramresponses for wild type ERManI, E330Q, and T688A (supple-mental Fig. 3) are representative of the effects of temperatureon the respective enzymes. Because of the fast on- and off-rates,binding to the wild type enzyme could only be measured at theequilibrium plateau values for the sensorgrams, and below15 °C there was little effect of increasing temperature (supple-mental Fig. 3). At higher temperatures there was a progressivedecrease in the sensorgram amplitude, presumably as a resultof a combination of binding and hydrolysis of the ligand andsubsequent release of the enzymatic product. For the T688Amutant, the off-rate progressively increased with increasing
temperature, whereas the equilibrium sensorgram values in-creased up to �20 °C and then progressively decreased (sup-plemental Fig. 3). We have interpreted these data to indicatethat glycan binding was more prevalent than glycan hydrolysisat low temperature but that increased hydrolysis at highertemperature led to an increased off-rate and an inflection forthe equilibrium sensorgram values. In contrast, the off-rate forthe E330Q mutant was considerably less influenced by increas-ing temperature, whereas the on-rate increased with temper-ature, leading to an increase in equilibrium binding (supple-mental Fig. 3). Similar results were revealed when the bindingdata were subjected to van’t Hoff analysis, where the slopes ofthe plots indicated that the temperature dependence of the KD
for the T688A mutant was greater than for the E330Q mutant(supplemental Fig. 1B). Eyring analysis demonstrated that thetemperature dependence of the KD for the T688A mutant wasmainly due to an increase in the dissociation rate with increas-ing temperature (supplemental Fig. 1, C and D). Calculation ofthe thermodynamic parameters for the glycan association anddissociation (supplemental Table I) indicated that there was asignificantly greater entropic contribution to glycan dissocia-tion for the T688A mutant by comparison to the E330Q mu-tant, as would be predicted for the greater temperature de-pendence of dissociation.
To confirm the temperature dependence of enzyme activityunder the conditions used for SPR analysis (pH 7.0) we com-pared the specific activities of the various enzyme forms at 10and 37 °C at this pH. The wild type enzyme had a 30-foldincrease in specific activity between 10 and 37 °C, whereas theT688A mutant increased 10-fold and the E330Q mutant in-creased only 2-fold over the same temperature range (data notshown). Because the E330Q mutant has a reduced pH optimumfor catalysis (pH 5.3) relative to the wild type enzyme (pH 7.1)(46), we also tested the pH dependence of glycan binding by theE330Q mutant. At pH values �7.0, the E330Q mutant exhibitshigh affinity binding (KD 1 �M) (Table III and supplementalFig. 4), whereas at pH values below 7.0 both the on- andoff-rates are altered to result in an enzyme with a 46-fold lowerbinding affinity at pH 5.0. We interpret the alterations in on-and off-rates and decrease in glycan affinity to reflect an in-crease in glycan hydrolysis under conditions closer to the pHoptimum of the mutant enzyme. These data indicate that theE330Q mutant is an effective model for binding analyses underour conditions for SPR (pH 7.0, 10 °C) because, in contrast towild type ERManI or the T688A mutant, there are minimalcontributions of ligand hydrolysis.
TABLE IISummary of the SPR binding affinity data for wild type and mutant forms of ERManI
Ligand Protein Fitting typea ka kd KD
KD
KD (wt)
s�1�M�1 � 1000 s�1 � 0.001 �M -fold
Man9GlcNAc2 glycopeptideb Wild type SS 48.6 � 1.7 1Wild type (�Ca2�)c 1:1 1.18 � 0.16 90.5 � 16.3 76.4 � 3.8 1.6T688A 1:1 14.0 � 3.9 13.1 � 3.5 0.94 � 0.36 0.02F659A SS 194 � 80.6 4.0
a 1:1 refers to a 1:1 nonlinear Langmuir fit of the binding data to derive the ka, kd, and KD values from the kinetic SPR binding sensorgrams asdescribed under “Materials and Methods.” SS refers to a steady-state fit of the binding data from maximal equilibrium values of the SPRsensorgrams as described under “Materials and Methods.” For examples of each type of data fitting, see Fig. 2.
b Binding analyses were performed at 10 °C.c Immobilized surfaces were treated with EGTA overnight by continuous flow of running buffer containing 5 mM EGTA at 5 �l/min, and the
binding analyses were performed in buffer containing 200 �M EGTA.d ND, no detectable binding (KD �10 mM).e Binding analyses were performed at 25 °C.
Glycan Binding and Catalysis by �-1,2-Mannosidases 29843
Energetic Contributions of Glycan Binding to the E330QMutant of ERManI—The E330Q mutant of ERManI was usedas a model to investigate the contributions of various resi-dues in a Man9GlcNAc2 oligosaccharide for binding to themutant enzyme. Individual Man9GlcNAc2, Man8GlcNAc2,Man6GlcNAc2, and Man5GlcNAc2 glycans, as well as mannosedisaccharides (Fig. 3A), were examined for their respectivebinding parameters (Table IV), and the resulting KD valueswere converted to their corresponding �G values for binding.The contributions of individual residues or groups of residueswere subsequently calculated by a combination of differencecalculations (��G values) using the rationale shown in Fig. 3Band Table V. These calculations indicate that binding of theglycone residue in the �1 subsite contributes only �21%(�1.68 kcal/mol) to the overall binding energy of aMan9GlcNAc2 ligand (Table V and Figs. 3C and 4), whereas the�1 subsite residue (M7 in Figs. 1E and 4) contributes �52%(4.2 kcal/mol) to the overall binding energy. Residues M9, M8,and M11 (Fig. 1E) together contribute �19% (1.53 kcal/mol) tothe glycan binding energy, whereas the remainder of the glycancontributes �0.42 kcal/mol (�6%), and the peptide backbonecontributions are negligible (�0.19 kcal/mol) (Fig. 3C andTable V).
DISCUSSION
Class 1 (CAZy glycosylhydrolase family 47 (25–28)) �-man-nosidases play key and diverse roles in glycan maturation anddisposal of misfolded glycoproteins in the ER (2, 9, 24, 32–36,38). Prior studies have revealed that the overall protein fold forthe catalytic domain of members of this family is an (��)7barrel structure with a catalytic site in the core of the barrel(41–45). Co-complex structures between ERManI and dMNJ,Kif (42), or a mannobiose thiodisaccharide (46) revealed theconformational itinerary of the glycone during catalysis. Dis-tortion of the glycone into a 3S1 conformation during substratebinding has been proposed to predispose the substrate for hy-drolysis by a least motion conformational twist through a ring-flattened 3H4 transition state producing an inverted enzymaticproduct in a 1C4 conformation (46). Several questions remainregarding the energetics of substrate binding during catalysisand the nature of the active site chemistry. Conservation in thepositions of key residues in the �1 and �1 enzyme subsitesindicates that both the ER and Golgi subfamilies of hydrolasescleave Man-�1,2-Man linkages by a similar mechanism. How-ever, the ER and Golgi enzymes recognize distinctive terminalbranches of Man9GlcNAc2 substrates. Prior structural data onputative enzyme-product co-complexes for both the ER andGolgi enzymes support the proposal that the respective en-zymes have distinctive geometries in the clefts leading from thebarrel cores that engage the � �1 residues of the respectivesubstrates and confer the unique branch specificities for thedifferent enzyme subfamilies (41). In an effort to understandthe energetics of substrate binding and catalysis for the class 1mannosidases, we have examined the interactions between the
wild type and mutant forms of ERManI by using various sub-strates and inhibitors.
Two residues that were shown previously to play roles in
FIG. 3. Strategy for determining the energetic contributions ofrespective Man9GlcNAc2 glycan residues for binding to theE330Q mutant of ERManI. A series of SPR binding studies with theimmobilized E330Q mutant enzyme was performed by using variousligand structures as indicated in A. In addition to Man9GlcNAc2-PA andMan9GlcNAc2-glycopeptide ligands, discrete isomers of Man8GlcNAc2-PA, Man6GlcNAc2-PA, and Man5GlcNAc2-PA, as well as Man�1,2Mandisaccharides, were tested. For each ligand, binding analyses wereperformed, and KD values were determined (Table IV). The resultantKD values were converted to values of �G (Table IV) by using therelationship �G �RTln(1/KD), and energetic contributions to glycanbinding were then calculated by a series of �G difference calculations(��G calculations) as shown in the example calculation (B). The calcu-lations for combinations of residues are summarized in Table V, and thedata are graphically summarized in C. The glycone residue was foundto contribute only 20.9% to the overall glycan affinity, whereas the �1subsite residue contributed 52.4% to the glycan binding energy.
TABLE IIISummary of the pH dependency of Man9GlcNAc2 binding to the E330Q mutant of ERManI
SPR binding studies were performed with the immobilized E330Q mutant enzyme at 10 °C at varied values of pH. At each pH the SPRsensorgram traces were obtained for the binding of Man9GlcNAc2 (supplemental Fig. 4), and values for ka, kd, and KD were determined. Minimalchanges in the sensorgram profiles were detected at pH 8–6, whereas the on-rates and off-rates were both considerably altered at pH 5.0.Calculated values for KD indicated a progressive 40-fold lowering of glycan binding affinity between pH 8 and 5.
binding to � �1 subsite residues (46, 61) were examined fortheir effects on catalysis and substrate binding. The R461L,R461A, and R597A mutants all resulted in reduced catalyticrates (varying 6–34-fold) and slightly increased Km values (3–5-fold) (Fig. 4). Both the R461A and R597A had binding affin-ities for dMNJ and Man9GlcNAc2 that were comparable withthe wild type enzyme, whereas the R461L mutant had nodetectable Man9GlcNAc2 binding (KD �1 mM). Arg461 has beenproposed to play a major role in ERManI substrate bindingand recognition (61), contrasting with a Leu at the equivalentposition for the Golgi enzymes. Only the R461L mutant wasfound to readily catalyze the cleavage of Man9GlcNAc2-PA toMan8–6GlcNAc2-PA, similar to data reported for S. cerevisiaeERManI (61). Our working hypothesis is that Arg461 contrib-utes to high substrate binding affinity and substrate specificityonly within the context of the overall geometry of the ERManIglycan binding cleft. The R461L mutation would eliminatespecific hydrogen bonding interactions that confer glycan affin-ity and specificity as well as creating space at the core of theglycan-binding site for more flexible interactions with alterna-tive terminal glycan branches. However, the R461A mutantwould be predicted to allow even greater flexibility for glycanbinding, yet the latter mutant maintains the restricted sub-strate specificity of wild type ERManI. These data suggest thatthe Leu residue at this position provides a positive role inbroadening the substrate specificity, but within the inappro-priate context of the ERManI glycan-binding cleft steric con-straints preclude high affinity glycan interactions. In contrast,within the context of the active site clefts of the Golgi subfamilyof enzymes, the corresponding Leu residue would be expected
to confer high affinities of substrate binding and appropriatebranch specificities for the latter enzymes.
Initial studies of inhibitor binding examined the energeticsand kinetics of interactions by ITC and SPR. Both approachesindicated that binding of the inhibitors was highly exothermic,and the KD values from ITC were in close agreement with Ki
values from catalytic measurements and binding affinities bySPR (supplemental Table II). The major difference in structurebetween dMNJ and Kif is the fused five-membered ring in thelatter compound (Fig. 1F) that causes Kif to be “pre-loaded” inthe high free energy 1C4 conformation prior to binding to theenzyme (46). The restricted conformation and the additionalinteractions between the enzyme and the Kif five-memberedring were proposed to account for the higher binding affinity ofKif in comparison to dMNJ (42). Surprisingly, binding studiesby SPR indicated that the on-rate for binding to wild typeERManI was comparable between dMNJ and Kif, but the off-rate for Kif was considerably slower. Thus, the restricted con-formation of the Kif six-membered ring did not accelerate bind-ing of the inhibitor but significantly slowed dissociation. SPRbinding studies between Kif and the F659A mutant, whichshould eliminate the van der Waals interactions with the C4-C5-C6 region of the ring-constrained inhibitor, significantlyincreased the off-rate and lowered the overall inhibitor bindingaffinity. Consistent with the lower binding affinity for Kif tothe mutant enzyme, binding of dMNJ to the F659A mutant wasnot even detectable by SPR (KD �10 mM). These data indicatethat Phe659 plays a critical role in stabilizing the glycone in theactive site and that the van der Waals interactions with thering-constrained inhibitor contributes to its slow dissociationrate from the active site. By extension, the greater ring flexi-bility of dMNJ or the substrate/product glycone residue asso-ciated with the wild type enzyme would be predicted to con-tribute an entropic component favoring dissociation from theactive site. Consistent with a role for Phe659 in inhibitor bind-ing, the F659A mutant also caused a 123-fold reduction in kcat,suggesting that interactions between Phe659 and the glycone inthe �1 subsite play a significant role in constraining the sub-strate into the 3S1 conformation required for catalysis.
At the base of the �1 subsite a protein-bound Ca2� ioninteracts directly with the glycone 2�- and 3�-hydroxyl residues.The ion is coordinated directly with the O-� and carboxyl oxy-gens of Thr688 and indirectly with four water molecules asso-ciated with Glu residues in the core of the �1 subsite. Twotypes of studies examined the roles of the Ca2� ion in glycanbinding and catalysis. First, SPR binding studies on the Ca2�-depleted enzyme indicated that the ion contributes to glycanon- and off-rates, but the enzyme had an almost identicalequilibrium binding affinity for the Man9GlcNAc2 ligand as theenzyme containing bound Ca2�. Binding of dMNJ was drasti-cally reduced, with a predominant effect on reducing the on-rate of the inhibitor. Second, altering the Thr688 side chain to
TABLE IVSummary of oligosaccharide binding interactions to the E330Q mutant of ERManI
a Schematic representations of the glycan ligand structures are shown in Fig. 3A.b �G283 �RTln(1/KD), at 283 K and R 1.987 cal � mol�1 � degree�1.c -PA refers to pyridylamine-tagged oligosaccharides used as ligands in the SPR binding studies.d -GP refers to purified glycopeptides from soybean agglutinin as ligands in the SPR binding studies.
TABLE VCalculation of binding energy contributions for various glycan
components to the E330Q mutant of ERManI establishing the �1subsite residue as the major contributor to glycan binding
Free energy calculations were generated by a strategy shown in Fig.3B using a color convention for the residues in the high mannoseligands as indicated in the same figure. ��G calculations to define theenergetic contributions of individual or combinations of residues wereperformed by calculating the differences in free energies of binding forvarious glycan ligands (from Table IV) to the E330Q mutant.
Glycan Binding and Catalysis by �-1,2-Mannosidases 29845
an Ala did not significantly alter Ca2� binding affinity for theenzyme, but it reduced kcat by 61-fold and increasedMan9GlcNAc2 binding affinity by 50-fold. These data suggestthat alterations in the �1 subsite can lead to an increasedbinding affinity for an uncleaved substrate. The data also sug-gest that glycone interactions with the enzyme-bound Ca2� ion,in the context of the appropriate tethering by Thr688, directlyfacilitate catalysis.
One of the main goals of our studies on ERManI was to mapthe energetics of interaction between glycan substrates and theactive sites of class 1 mannosidases and to identify whichresidues contribute to catalysis and substrate specificity formembers of the different enzyme subfamilies. The strategy forthese studies will be to examine the individual contributions ofenzyme residues in their interactions with the glycan sub-strates and the individual contributions of glycan resides intheir interactions with the enzyme. The wild type enzyme is notan effective model for SPR binding studies of this type, becausethe rapid on- and off-rates have contributions from both bind-ing and ligand hydrolysis. A preferable model would be anenzyme form that is compromised in hydrolysis yet maintainssubstrate binding with similar characteristics to the wild typeenzyme. For maximal utility, the mutant enzyme would alsohave high glycan binding affinities but relatively slower on-and off-rates than the wild type enzyme so that subtle changesin association and dissociation rates could be readily measured.The E330Q mutant appears to fulfill all of these criteria, be-cause it has minimal catalysis under the conditions that weroutinely use for SPR binding studies (10 °C, pH 7.0), yet itretains high affinity glycan binding with reduced on- andoff-rates. As a demonstration of the utility of this mutant,we examined the binding affinities of a collection of glycan
ligands, and we used the relative binding energies to calculatethe contributions of the respective glycan residues in aMan9GlcNAc2 substrate for their interactions with the mutantenzyme. The method yielded reproducible results, because thebinding energies calculated from different combinations of gly-cans yielded reasonably similar energetic contributions (TableV and Fig. 3).
The results of the glycan binding studies revealed that the�1 subsite residue (residue M7) contributes a majority (�52%)of the binding energy for a Man9GlcNAc2 ligand, whereas theglycone binding to the �1 subsite contributes only �21% of thebinding energy. Another �19% of the binding energy is con-tributed from the other peripheral �1,2Man residues, and�7.6% of the binding energy comes from core glycan residues.These data are consistent with binding data obtained from therespective ERManI mutants (Fig. 4). Mutations in the �1subsite or conditions that significantly reduce dMNJ bindingaffinity or catalysis either have a minimal effect onMan9GlcNAc2 glycan binding (F659A mutant or Ca2� deple-tion) or actually increase glycan binding affinity (T688A andE330Q mutants). Thus, the contributions of the ��1 subsiteresidues to the overall binding affinity can compensate forcompromised interactions with glycan substrates in the �1subsite (Fig. 4).
In contrast, alterations in the �1 subsite, such as the D463Nor R461L mutants (Fig. 4), abolished glycan binding (KD values�1 mM). The former side chain anchors the interactions be-tween the enzyme and the M7 residue in the �1 subsite byhydrogen bonding with the sugar O-3� and O-4� hydroxyls (46).For the latter residue, Arg461 has been shown to form a matrixof interactions with mannose residues M7, M4, and M3 (43, 61),yet substitution with an Ala residue (R461A) resulted in a near
FIG. 4. Summary of the enzyme kinetics and binding studies for key protein and glycan residues in the ERManI active site. Aschematic display of the catalytic and binding components in the ERManI active site is shown highlighting the residues interacting with the �1,�1, and �2 subsite glycan residues. Fold differences for kinetic parameters (values for kcat and Km) and SPR binding parameters (KD values forbinding to Man9GlcNAc2 and dMNJ) relative to values for wild type ERManI for each mutant are indicated adjacent to each respective amino acid.In addition, the respective proposed function for each residue based on the binding and kinetic analyses is indicated adjacent to each amino acidin red text. Energetic contributions to binding within the �1 and �1 subsites are also indicated by the dotted lines with shading. The “Discussion”summarizes the data from the kinetic and binding analyses for the wild type and mutant enzymes and describes their respective contributions tosubstrate binding and catalysis.
Glycan Binding and Catalysis by �-1,2-Mannosidases29846
wild type binding affinity for Man9GlcNAc2. In contrast, theR461L mutant has no detectable Man9GlcNAc2 binding, con-firming that there is likely a problem with steric hindrance inthe latter mutant.
In conclusion, the combined studies on catalysis and glycanbinding by wild type and mutant ERManI forms revealed anactive site cleft that promotes substrate binding predomi-nantly through interactions with the � �1 subsite residues(Fig. 4). How do these data compare with our emerging modelfor catalysis by class 1 �-mannosidases (46)? The docking ofthe glycone residue in a high free energy 3S1 conformation,predisposed for glycoside bond hydrolysis, is partly facili-tated by van der Waals interactions with Phe659 and addi-tional hydrogen bonding to �1 subsite residues. However, thepredominant source for substrate binding energy is providedby interactions with the �1 residue, dominated by the pair ofhydrogen bonds from Asp463 to the 3�- and 4�-hydroxyls of the�1 subsite residue (Fig. 4). These latter interactions likelyoffset the entropic penalty for binding to a high free energyglycone conformation in the �1 subsite, creating favorableenergetics for the conformational distortion required for gly-coside bond hydrolysis. Additional binding energy and branchspecificity are provided by the interactions with the � �1subsite residues. Interactions between the glycone residueand the enzyme-bound Ca2� ion influence the association anddissociation rates for glycan substrates, but do not signifi-cantly increase their respective equilibrium binding affini-ties. However, the Ca2� ion does promote catalysis, presum-ably through the assistance of Thr688, the sole residueinvolved in direct coordination with the divalent cation. Inthe absence of the Thr688 side chain, a solvent water moleculelikely replaces the lost point of Ca2� ion coordination. Ca2�
binding affinity is not reduced in the T688A mutant, yet thecatalytic rate is significantly reduced, and glycan bindingaffinity is surprisingly increased. These data strongly sug-gest that Thr688 aids in Ca2�-mediated catalysis, eitherthrough appropriate positioning of the Ca2� ion in the activesite or by providing appropriate electrostatics to the divalentcation. It is worth noting that the water nucleophile in theinverting catalytic mechanism is directly coordinated tothe Ca2� ion (46), and an isosteric amide substitution of theadjoining general base residue (E599Q) reduced catalysis by13,000-fold (Fig. 4) (46). However, this mutation did notincrease the binding affinity for the uncleaved substrate.Thus, altering the electrostatics of the general base functionfor activation of the water nucleophile does not account forthe increased glycan binding affinity for the T688A mutant.An alternative role for the Thr688 side chain could be amechanical tethering of the Ca2� ion in a favorable positionadjacent to the �1 subsite that is required for efficient catal-ysis. A similar effect of reduced catalysis and increased bind-ing of an uncleaved substrate is found for the general acidmutant E330Q. Future studies on the structures of co-com-plexes between the T688A and E330Q mutants with un-cleaved substrates or substrate analogs should provide in-sights into the roles of the glycone conformational changesand the enzyme-associated Ca2� ion in glycan hydrolysis.
The use of the E330Q mutant in SPR binding studies wasalso shown to be an effective tool in assessing the bindingcontributions of respective residues within the Man9GlcNAc2
substrate. Applying a similar approach to map the contribu-tions of oligosaccharide substrate residues for the other Class 1mannosidases will reveal the molecular basis of substrate rec-ognition and specificity for this diverse enzyme family. Moreimportantly, the ability to measure detailed binding affinitiesand kinetics using the equivalent of the E330Q mutant as a
model should provide critical information for the analysis ofnew selective inhibitors for class I mannosidases as potentialtargets for human protein misfolding disorders.
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Glycan Binding and Catalysis by �-1,2-Mannosidases29848
Khanita Karaveg and Kelley W. MoremenQuality Control
-Glycan Processing and Endoplasmic ReticulumN-Mannosidases Involved in α47) Energetics of Substrate Binding and Catalysis by Class 1 (Glycosylhydrolase Family
doi: 10.1074/jbc.M505130200 originally published online May 23, 20052005, 280:29837-29848.J. Biol. Chem.
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