Human acid fl-glucosidase: use of sphingosyl and N-alkyl-glucosylamine inhibitors to investigate the properties of the active site

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1 12 Bichimica et Biphysica Acta, 1039 (1990) Elsevier BBAPRO Human acid fl-glucsidase: use f sphingsyl and N-alkyl-glucsylamine inhibitrs t investigate the prperties f the active site Paul Greenberg 1, Alfred H. Merrill 2, Dennis C. Litta 3 and Gregry A. Grabwski 1 1 Divisin f Medical and Mlecular Genetics, Department f Pediatrics, Munt Sinai Schl f Medicine, New Yrk, NY and the Departments f e Bichemistry and 3 Chemistry, Emry Uniersity, Atlanta, GA (U.S.A.) Received 13 December 1989) Key wrds: Sphinglipid; Transitin-state analgue; Enzyme inhibitr; fl-glucsidase; (Human) Human acid fl-glucsidase (D-glucsyl-N-acylsphingsine gluchydrlase, EC ) cleaves the fl-glucsidic bnds f glucsyiceramide and synthetic fl-glucsides. The specificity f binding t the active site f this enzyme was evaluated using series f inhibitrs including synthetic sphingsines, N-alkyl(Cn)-dexynjirimycins (l,5-didexy-5-iminglucse) and N-Cn-glucsylamines. The sphingsines were rapidly reversible inhibitrs with maximal ptency (IC~ pm) at chain lengths f carbns. The presence f unsaturatin between C4 and C5 was required fr inhibitin f enzyme activity. Neither the nature f this bnd (duble r triple bnd) nr the presence f erythr r thre cnfiguratins at C2 influenced inhibitry ptency. The N-C10- t N-C14-dexynjirimycins were rapidly reversible inhibitrs with K i ~ nm. In cmparisn, the 1-amin glucse derivatives, i.e., N-C~-glucsylamines (n ), were mre ptent (IC nm) and their maximal inhibitry ptencies were dependent n time as well as enzyme and suhstrate cncentratins: i.e., the N-Ctz- t N-C ms-glucsylamines were cmpetitive, slw-tight binding inhibitrs. Analyses f prgress curves at varius N-Cn-glucsylarnine (n ) cncentratins indicated the frmatin f rapidly dissciating initial E1 cllisn cmplex which then underges a cnfrmatinal change t a slwly reversible El* cmplex. These results were cnsistent with the lng chain N-C~-glucsylamines being reactin intermediate analgues and with this enzyme's hydrlytic mechanism requiring a cnfrmatinai change during the transitin state. Intrductin Human acid fl-glucsidase (D-glucsyl-Nacylsphingsine gluchydrlase, EC ) is a membrane-assciated lyssmal enzyme which cleaves the fl-glucsidic linkage f its majr natural substrate, glucsylceramide as well as synthetic fl-glucsides [1,2]. The defective activity f this enzyme results in the variants f Gaucher disease, the mst prevalent lyssmal strage disease [3]. The cmplete amin acid sequence (M r = 55750) f this glycprtein has been deduced frm the cdna [4,5]. Based n studies f the Abbreviatins: N-Cn-glucsylamines r-dexynjirimycins, N-alkyl derivatives with specified chain lengths (Cn, n = 6-18); Cn-sphingsine, sphingsine derivatives f n =11-20 carbns in chain length; 4MU-Glc, 4-methylumbelliferyl-fl--glucpyranside; ICs0, cncentratin f inhibitr which results in 50% lss f initial enzymatic activity. Crrespndence: G.A. Grabwski, Munt Sinai Schl f Medicine, One Gustave Levy Place, New Yrk, NY 10029, U.S.A. ph variatin f enzyme activity and the effects f reversible inhibitrs, e.g., N-hexylglucsylsphingsine [6], an active site was suggested t cntain tw inizable grups which participate in substrate catalysis [6,7]. Recently, active site residues have been lcalized t the carbxy ne-third f the acid fl-glucsidase sequence using suicide substrates, cnduritl B epxide derivatives, as affinity labels [8]. These studies als identified Asp 443 as a carbxylate which functins as a nuclephile fr catalysis [8]. Acid fl-glucsidase requires detergents, negatively charged lipids and/r a naturally ccurring prtein effectr, a 'c-fl-glucsidase', fr ptimal hydrlysis f glucsylceramide r synthetic substrates [6-13]. Analyses f the inhibitry effects f extensive series f substrate analgues, as well as glucse derivatives and epimers, indicated that the active site cntained subsites fr interactin with the fatty acid acyl, sphingsyl and glycn mieties f glucsylceramide substrates [7,13,14]. The affinity f substrate analgues fr the active site was suggested t be mdulated by the chain length and cmpsitin f the sphingsyl miety as well as the cnfiguratin and substituent grups f /90/$ Elsevier Science Publishers B.V. (Bimedical Divisin)

2 13 the glucpyranse ring [17]. In particular, large increases in ptencies f inhibitin were bserved with glucse derivatives having amin grups n C1 r C3, r a 5-imin grup [7]. With the 5-imin glucse derivatives and the very ptent N-alkyl-dexynjirimycin (1,5-didexy-5-iminglucse) derivatives [7,12], the variatins f inhibitry ptencies with ph were cnsistent with the unprtnated frm f the inhibitr binding t a mnprtnated enzyme frm [7]. Hwever, inferences as t the catalytic mechanism were nt clear, since the N-alkyl-dexynjirimycin inhibitrs did nt have the prperties f analgues f reactin intermediates during substrate hydrlysis [7,13]. T further investigate requirements fr interactins at the active site and t gain insights int the reactin mechanism, sphingsine and N-alkyl(C,)-glucsylamine derivatives f varius chain lengths and cmpsitins were synthesized and their inhibitry ptencies determined using hmgeneus acid fl-glucsidase purified frm human placenta. The results f these studies prvide additinal supprt fr mdulatin f substrate affinity by the structure f the sphingsyl miety. In additin, the lng chain N-C:glucsylamines were fund t be slw-tight binding inhibitrs with prperties cnsistent with their being analgues f reactin intermediates. Materials and Methds Materials The fllwing were frm cmmercial surces: A1- kylarnines (hexyl- (C5), ctyl- (C8), decyl- (C10), ddecyl- (C12), tetradecyl- (C14), hexadecyl- (C16), and ctadecyl- (C18)) and (1S,2S)-2-amin-l-phenylprpane- 1,3-dil frm Aldrich, Milwaukee, WI. Sdium taurchtate (Calbichem, Ls Angeles, CA), bvine brain phsphatidylserine (Avanti Plar Lipids, Pelham, AL), t-glucse and 4-sphinganine (dihydrsphingsine) (Sigma, St. Luis, MO), 4-methylumbelliferyl-fl-Dglucpyranside (4MU-Glc) Genzyme, Bstn, MA. Other reagents were reagent grade r better. The fllwing were prepared and purified as described: Natural sphingsine f mixed chain lengths [15]. The synthetic sphingsine derivatives [17] and flglucsylamine [18] were prepared as described. N- alkyl(cn)-giucsylamines were synthesized [16], recrystallized frm methanl, lyphilized and stred dry under vacuum until used. Melting pints fr the respective N-C:glucsylamines derivatives were: N-C 6- (lit. = C, m.p. = C); N-Ca- (lit. = 102 C, m.p. = C); N-C10- (lit. = C, m.p. = C); N-C12- (lit. = C, m.p. = C); N-C14- (lit. =104 C, m.p.=104 C); N-C16- (lit.= 103 C, m.p. = 104 C); N-C~s- (lit. = C, m.p. = I04 C). N-decyl- (C10), N-ddecyl- (C12), N-tetrade- cyl- (C14) dexynjirimycins were generus gifts frm Dr. Gunter Legler. Enzyme surces Human placental acid fl-glucsidase was purified by affinity chrmatgraphy [19]. Hmgeneity f this preparatin was demnstrated by the presence f a single N-terminal amin acid sequence which crrespnded t authentic acid fl-glucsidase. The maximal specific activities (4 mm 4MU-Glc substrate) f the uninhibited enzyme were nml/h per mg prtein in 0.04 citrate/phsphate (ph 5.7) cntaining 1.0% taurchlate and nml/h per mg prtein in 0.2 M acetate (ph 5.8) cntaining 20 #g/ml f phsphatidylserine. The Vma x vs. ph prfiles were brad with the enzyme having abut 70% f maximal specific activity at ph 6.5 in either f the abve reactin mixtures. The enzyme cncentratin was calculated frm the prtein cncentratin [24] and the mlecular weight (unglycsylated) f as calculated frm its amin acid sequence [4,5,19]. Enzyme assays Except fr the N-C:glucsylamines which were stred dry under vacuum, lipidal cmpunds were made as highly cncentrated stck slutins in chlrfrm/methanl (v/v, 2: 1) and stred at -20 C under nitrgen. The slvents in aliquts f these slutins were evaprated t dryness at rm temperature under nitrgen and then dispersed int aqueus buffers by snicatin until the slutins were clear. Aqueus dispersins f phsphatidylserine were prepared and used n the same day r were kept under nitrgen until use. Except where nted, assays were cnducted, using 4 mm 4MU-Glc as the substrate, by cntinuus mnitring f increasing flurescence intensity due t prduct, 4-methylumbelliferne, frmatin using excitatin and emissin wavelengths f 360 and 460 nm, respectively. Althugh greater flurescence yields (abut 8-fld) f 4-methylumbelliferne culd be btained at an excitatin wavelength f 320 nm, substantial flurescence quenching was bserved with the substrate, 4MU-Glc, at high cncentratins [20,21], i.e., between 4 and 16 mm. This was minimized by using the excitatin wavelength f 360 nm. The temperature was maintained at 25 C in a temperature cntrlled cell. All samples and slutins were equilibrated at 25 C prir t initiatin f the reactins. Data frm prgress curves were analyzed by fitting t the apprpriate nn-linear r linear mdels (see Eqns. 1-7 in Results) using the Systat TM statistical prgrams. Initial estimates f kinetic values fr these regressin analyses were btained graphically [22]. The enzymatic activity was shwn t be stable under all assay cnditins by determinatins f activity at several times during the curse f the experiments n any day. Quantitative end-pint assays f

3 14 uninhibited enzyme activity verified that less than 5% f the 4MU-Glc was cnsumed under all assay cnditins. Prgress curves were btained using tw different methds: (1) Enzyme and inhibitr, which had been thermally equilibrated at 25 C, were added separately but simultaneusly t buffered slutins ( /~1) cntaining the required cncentratins f 4MU-GIc and negatively charged lipids at 25 C. Thrugh mixing was achieved rapidly (< 4 s) by careful, manual upand-dwn agitatin in situ in the temperature equilibrated cuvette with a plastic L-shaped spatula. The pure enzyme stck was diluted s that aliquts cntained the required final cncentratins f buffer and reagents in the final assay slutin. The inhibitrs were added as slutins in methanl r ethanl. (2) Enzyme, diluted in the apprpriate buffers, was incubated with cncentrated inhibitr fr min prir t additin t the substrate slutin. The final enzyme and inhibitr cncentratins in the substrate slutin were calculated t duplicate thse utlined abve in the first methd. This methd will be termed the 'preincubatin' methd. Fr either methd, the ethylene glycl cncentratins prir t additin t substrate slutins were less than 1% and the inhibitrs were added t the enzyme in methanl r ethanl (0.5-3/~1) which had n effect n enzyme activity. The reactin vlumes fr all prgress curves were 1.0 ml. The sphingsyl and N-C,-dexynjirimycin derivatives were rapidly reversible inhibitrs within the time frame f these experiments. The ICs0 values fr the sphingsine derivatives were determined by end pint assays r frm prgress curves with essentially identical results. The ICs0 values fr the N-C,-glucsylamines and the N-C,-dexynjirimycins were determined by prgress curves. ICs0 values crrespnd t the cncentratin f inhibitr which resulted in a 50% decrease f the uninhibited (cntrl) acid fl-glucsidase activity. Since hydrlysis f glucsylceramide cannt be mnitred cntinuusly, pilt, shrt-term (5-10 rain) end pint assays were cnducted using flurescent glucsylceramide as substrate [23]. Althugh highly reliable assays culd nt be btained during the first 5 rain f reactin, flurescent ceramide prductin between 5-10 min indicated the same degree f inhibitry ptency f the N-Ca2- r N-C16-glucsylamines tward glucsylceramide r 4MU-Glc hydrlysis. Results Inhibitin f acid fl-glucsidase by sphingsine derivatives The ICs0 values btained with varius sphingsine derivatives in a taurchlate based system are shwn in Table I. Very similar ICs0 values were btained frm assays cntaining phsphatidylserine (20 #g/ml). The ptency f inhibitin f the 4-sphingenines (Fig. 1A) f chain lengths frm carbns varied ver a 4.5-fld TABLE I IC5 values fr sphingsme and N-alkyl-glycn inhibitrs f human acid fl -glucsidase Inhibitr IC50 value Sphingsine derivatives (#M) 4-sphingenine CI 1-4-sphingenine Cl4-sphingenine Cl6-4-sphingenine C18-thre-4-sphingenine Cls-4-sphing-yne-ne C20-4-sphingenine 245 :k 6 N-Me-C20-4-sphingenine sphinganine > N-acetyl-sphinganine > amin-l-phenyl-prpan-l,3-dil > N.Cn_Glucsylamine s 3 (nm) N-C 6 55 N-C s 12 N-Cl 1.9 N-C N-CI4 2.0 N-C N-C N.Cn.Dexynjirimycins 3 (nm) N-Cl 56 N-Ca2 23 N-C14 25 i Assays cnducted in 0.04 citrate/phsphate buffer (ph 6.5) cntaining 1.0% taurchlate at 25 C. 2 4-Sphingenine (natural sphingsine) was frm Gaucher disease spleen. 3 Values were reprducible within + 20%. IC50 values were determined fr the N-Cn-glycns frm velcities btained frm the apparently linear prtins f prgress curves as described in Methds and Materials. range. Maximal inhibitry ptency was btained with the 14 carbn derivative, whereas the 11 and 20 carbn derivatives were abut 3-fld less ptent. Cmpared t the natural r synthetic erythr analgues, the thre cnfiguratin at carbn 2 had n majr effect n inhibitry ptency. Similarly, the presence f a linear triple bnd (C18-4-sphing-yne-ne) instead f the natural trans duble bnd at C4-C5 had n majr effect n the IC50 value. Saturated sphingsine derivatives, 4-sphinganine and N-acetyl-sphinganine, did nt inhibit the enzyme. Similarly, (1S,2 S)-2-amin-l-phenyl-prpane-l,3-dil (Fig. 1B), a cmpund cntaining a plar head grup structure similar t that f sphingsine, had n inhibitry effect. N-Cn-glucsylamine instability and assay cnditins The majrity f experiments were cnducted at ph 6.45, since the N-Cn-glucsylamines were mre stable at this than mre acidic phs (see belw). Pilt experiments at ph 5.5, 6.0 and 6.5 revealed the same inhibitry characteristics f the N-Cn-glucsylamines, i.e., the N-

4 15 C HO--CHg-CH-- CH--CH=CH--(CH.) -- CH 3 OH NH OH n=s t 14 ] R = Methyl (Me) r H R ~/~ O'k~-- (CH2) n--- CH 3 HO'~H / n= t 17 H HO ~---" N-- (CH 2 ) n-- CH3 HO n=gt13 Fig. 1. Structural diagrams f sphingsine (A), N-C,-glucsylamine (C) and N-e,-dexynjirimycin (dnm)(d) derivates and 2-amin-1- phenyl-prpan-l,3-dil (B). The derivative shwn in (A) is 4-spingenine. 4-Sphinganine (dihydrsphingsine) and 4-sphing-yne-ne are saturated r cntain a triple bnd, respectively, at C4-C5. ddecyl- t N-ctadecyl- derivatives were slw-equilibrating inhibitrs with similar t]/2 values fr maximal inhibitin. The time-dependent degradatin rates fr the individual N-C,-glucsylamines were determined by the lss f their inhibitry ptency tward acid fl-glucsidase at 25 C and at several different phs, and in the presence r absence f taurchlate ( mm) r phsphatidylserine (0-40/x/ml). Fr these experiments, cncentrated aliquts (typically 1/~1) f a N-C,-glucsylamine in methanl were added with rapid mixing t the desired aqueus slutins (typically 3-4 ml) cntaining 4 mm 4MU-Glc and negatively charged lipids. Immediately and at timed intervals (2-5 min), aliquts (150 /xl) f the inhibitr/substrate mixture were remved and added with rapid mixing t the enzyme slutin (1.1 nm). Assays were terminated after 5 min as described [9]. Half-lives f degradatin were determined by pltting the degree f inhibitin btained at each time pint beginning with 5 rain and extraplatin back t zer time. The half-lives f the N-C,-glucsylamines were 8-10 min (ph 4.5), rain (ph 5.5) and min (ph 6.5) fr the N-C 8 t N-C~8-glucsylamines in the presence f absence f taurchlate r phsphatidylserine. The inhibitry ptency f N-C 6- glucsylamine was slightly mre unstable with half-lives f 6 (ph 4.5), 9 (ph 5.5) and 20 (ph 6.5) rain. In experiments where highly cncentrated aqueus slutins f N-C,-glucsylamines were allwed t degrade fr several hurs, all inhibitry effects were lst. This result indicated that the N-C,-glucsylamines degradatin prducts did nt inhibit acid /3-glucsidase in cncentratin at least 100-fld greater than in these studies. The cncentratins f the N-C,-glucsylamines in the figures r in the text d nt include crrectins fr their degradatin unless specified. Despite this instability, the prgress cuves (Fig. 2, A and B) at times greater than abut 5 rain appeared linear. Under cnditins f negligible substrate depletin, prlnged incubatins times (25-45 min) and/r at lwer ph values, the prgress curves fr the N-Clz- and N-C16-glucsylamines became increasingly cncave-up, due t the spntaneus degradatin f these inhibitrs (data nt shwn). In similar experiments with N-C~0-, N-C~2- r N- Ca6-glucsylamines, cncentrated aliquts f these inhibitrs in methanl were diluted (at least 1 : 2000) int slutins cntaining enzyme (1.1 nm) in the absence f substrate. Aliquts f these mixtures were then assayed immediately r at varius times by additin t substrate slutins. The fact that n difference in the half-lives fr degradatin were bserved indicated that these N- C,-glucsylamines were either nt substrates r were very pr substrates f acid B-glucsidase. Inhibitin f acid fl-glucsidase by N-C,-glycn derivatives The N-C,-glucsylamines (Fig. 1C) were very ptent inhibitrs f acid fl-glucsidase. The N-CI2- t N-C18- derivatives were nt rapidly reversible inhibitrs but rather exhibited a slw apprach t maximal inhibitin (Fig. 2A) and, in preincubatin experiments (see Materials and Methds), a slw recvery frm maximal inhibitin (Fig. 2B). As a typical example, maximal inhibitin by N-C16-glucsylamine (Fig. 2A) was achieved at greater than 2 min, after the initiatin f the reactins. This finding was mst easily appreciated at cncentratins f inhibitr which decreased hydrlytic rates by greater than 50% (i.e., nm). Fig. 2A als demnstrates that the initial slpes f the prgress curves (i.e., the initial velcities) were dependent n the inhibitr cncentratin. This result indicated that significant amunts f 'EI' cmplexes were present during the apprach t the 'steady state' inhibited velcity. In cmparisn, the recvery f enzymatic activity was slw (Fig. 2B) fllwing preincubatin with cncentrated inhibitr. These results were typical fr the N-Ca2- t N-C18-glucsamines. T determine the relative ptencies f the N-C,-glucsylamine derivatives, ICs0 values were btained by pltting the 'steady-state' velcities, i.e., slpes f the

5 - 16 iii (,3 z ttl 0 09 ILl E ::3 LL.J iii > LU if- fa(/ 160 I 1 2O n 3.75 nm 7. 5 y 10 nm / 1 60 ~ nm.25 nm ~z 120 punds in assays cntaining taurchlate (Table I) were slightly greater than thse btained in assays cntaining phsphatidylserine (0.7 and 0.5 nm fr N-C10- and N-C12-glucsylamines, respectively). The ptency f the inhibitin decreased by abut fld fr the N-C 8- and N-C 6 derivatives cmpared t that fr the N-Ctand N-C12-glucsylamines. Smaller decreases in IC50 values were fund with increasing chain length (Fig. 3, A and B). Hwever, this decrease in ptency may be nly apparent, since the time t achieve maximal inhibitin was linearly related t the alkyl chain length (Fig. 3C) and because the half-lives fr degradatin f the N-C12- t N-CIs- derivatives were similar. In additin t having the characteristics f slw binding inhibitrs, the N-Ca2- t N-Cas-glucsylamines were tight-binding inhibitrs, i.e., their IC50 values were in the same range as, and varied directly with, enzyme cncentratin. Fig. 4 shws the results btained, with N-C12-glucsylamine as a typical example. The slpe f the curve (IC50 vs. [Et] ) in the inset f Fig. 4 was 0.57, indicating that the IC50 value was equal t abut ne-half f the enzyme cncentratin. This result indicated that significant depletin f free inhibitr ccurred in the reactin mixtures due t the frmatin f 'EI' cmplexes, and was cnsistent with the variatin f initial velcities btained with increasing inhibitr cncentratins (see Fig. 2A). Althugh the slpes varied, a similar d rr 4-0 / 5.0 nm I0 B A ~0.1 / """ TIME (rain) Fig. 2. Prgress curves fr the hydrlysis f 4MU-GIc in the presence r absence f N-C]6-glucsylamine. In (A) the assays were initiated by the simultaneus additin and rapid mixing f enzyme and inhibitr int the substrate slutin. In (B) cncentrated enzyme and inhibitr were preincubated tgether fr 20 rain and then rapidly diluted int the substrate slutin. The final cncentratins f inhibitr in the assay mixtures are nted. All assays were cnducted at 25 C and cntained 4 mm 4MU-GIc, 0.2 M sdium acetate (ph 6.45), 20 #g/ml f phsphatidylserine and 1.1 nm purified human placental acid fl-glucsidase. tangents t the apparently linear prtins f the prgress curves, against inhibitr cncentratins. The slpes f the linear prgress curves fr the uninhibited enzyme were the cntrl hydrlytic rates. In either taurchlate r phsphatidylserine based assay systems, the IC5 values were dependent n alkyl chain length (Table I, Fig. 3, A and B). The N-Ct0- and N-C12- derivatives were the mst ptent. The IC50 values fr these crn- <._/ n- i, ALKYL CHAIN LENGTH [INHIBITOR] (nm) Fig. 3. Inhibitry ptency f N-C,-glucsylamine derivatives. In (A), the 'steady state' hydrlytic rates were determined frm the linear prtins f prgress curves: e.g., Fig. 2, in the presence and absence f varius cncentratin f inhibitr. The curves fr the N-C 6- and N-Cs-glucsylamines were truncated t accmmdate the insets. Inset (B) shws the relatinship f I/ICs0 nm-1 and alkyl chain length. Inset (C) demnstrates the direct relatinship f time t achieve maximal inhibitry effect (designated inhibitin time) t alkyl chain length at 10 nm inhibitr. Assay cnditins were as in Fig. 2.

6 17 100' 80 _ 0.8 II\ If,,',,,, _~ ~<60.2nM 0 - \ \ ~ [INHIBITORI (nm) Fig. 4. Dependency f h~.jbitry ptency (ICs0) n acid fl-ghicsidase cncentratin. In the main figure, the 'steady state' hydrlytic rates were determined as in Fig. 3. The enzyme cncentratins are nted next t each curve. The ICs0 values frm the mean figure are shwn in relatinship t the enzyme cncentratin in the inset. The slpe f the curve in the inset was Assay cnditins were as in Fig. 2. dependency f ICs0 values n [Et] was btained with the N-C14- t N-Cls-glucsylamines under all assay cnditins. Because f these slw-tight binding prperties f the N-C,-glucsylamines, the standard steady-state studies t determine the nature f the inhibitin were nt applicable. Hwever, the similarity f the N-C,-glucsylamine structures t that f substrates and the linear variatin f ICs0 values with substrate cncentratin (Fig. 5) suggested that they exerted their effects at the active site. If cmpetitive inhibitin is assumed, the equatin btained by linear regressin f the data fr N-C12-glucsylamine (Fig. 5) prvides an estimate f the verall inhibitin cnstant, Ki* app, in the presence f 1.1 nm enzyme and taurchlate, since IC5 = Ki* (1 + [S]/K m ) (1) and frm Fig. 5 ICs0 = IS]. (2) Cnsequently, as [S] appraches zer, the ICs0 = Ki* rim. The cmparable result in the presence f phsphatidylserine (20 /ag/ml) was Ki* nm. Similar values were btained fr N-Ca0-glucsylamine, an inhibitr which was rapidly reversible n the time scale f these experiments. Frm Fig. 3C, the time required t apprach maximal inhibitin increased directly with alkyl chain length f the N-C,-glucsylamines. Extraplatin f this curve indicated that a N-Ctl- derivative wuld be predicted t be rapidly reversible and, indeed, the N-Cm-, N-C 8- and N-C6-glucsylamines as well as the parent cmpund, fl-glucsylamine (K i = 0.4 mm, data nt shwn), were rapidly reversible inhibitrs within the time scale f these experiments. The N-C,-dexynjirimycin derivatives (Fig. 1D), which are structurally similar t the N-C,-glucsylamines (Fig. 1C), were less ptent (Fig. 6 and Table I), rapidly reversible (Fig. 6) and nt tight binding inhibitrs. The prgress curves which were initiated by additin f the enzyme r frm 'preincubatin' experiments, were linear at all cncentratins f N-C,-dexynjirimycins. T mechanistically accunt fr the slw binding prperties f the lnger chain N-C,-glucsylamines and t estimate their kinetic cnstants, data frm prgress curves at several cncentratins f the N-C14- t N-C16- and N-Cas-glucsylamines were subjected t regressin analyses using the fllwing equatins [22]. These describe reactins which were initiated by the simultaneus additin f inhibitr and enzyme t substrate slutins: P= st + ( V - Vs)(1-e-*t)/k O s = VS/[ Kin(1 + I//Ki * ) + S] k = k 6 { [1 + (I/Ki* (1 + S/Km)]/[1 + (I/Ki(1 + S/Km) ] } ksk 6 = ( Ki/Ki* )-I k 3 k5 E~EI~EI* k4 k6 Scheme I where P, vs, v 0 and k are the prduct frmed (flurescence change), 'steady state' velcity, initial velcity and the apparent first-rder rate cnstant, respectively. V, S and K m were Vma x, substrate cncentratin and dissciatin cnstant ( mm) fr the 4MU Glc substrate. K i is the dissciatin cnstant fr the E1 cmplexes whereas Ki* and ks/k 6 are the verall dissciatin cnstant and the rati f the frward and reverse rate cnstants fr the El* cmplex (see Scheme I), respectively. The kinetic values (Table II) verestimated the actual values since n crrectins were made fr inhibitr degradatin and data frm prgress curves up t 10 min were required t estimate % Applicatin f Eqns. 3-6 prvide reasnable estimates f kinetic values [22] under the cnditins where 2[Et] < I t > Ki* (1 + S/Km) (7) Fr these experiments, E t = 1.1 nm and I t >_ 5 nm fr each inhibitr and at all time pints. Fr N-CI4- t N-Cts-glucsylamines, the rati, Ki/Ki*, was greater than ne and its value increased directly with alkyl chain length. Furthermre, the verall inhibitin cn- (3) (4) (5) (6)

7 I I I I I ~ I I [4MU-GIc] (mm) Fig. 5. Relatinship f the inhibitry ptency (IC50) f N-C12-glucsylamine t substrate (4MU-GIc) cncentratin. The data were fitted t a linear mdel by regressin analyses. The equatin fr the shwn theretical curve was ICs = [S]. Assays were cnducted in 0.2 M sdium acetate (ph 6.45) cntaining 1% taurchlate and 1.1 nm enzyme. Six inhibitr cncentratins between 0 and 10 nm were used t generate prgress curves at each substrate cncentratin. The 'steady state' inhibited velcities were as in Fig O TABLE II Inhibitin values fr acid fl-glucsidase by N-C, -glycns 1 Inhibitr Ki 2 Ki. ks/k 6 (nm) (nm) (nm) Glucsylamines N-Cla-glucsylamine N-C 16-glucsy lamine N-C18-glucsy lamine Dexynjirimycins N-C12-dexynjirimycin N-Cl4-dexynjirimycin =5 =12 = 30 1 Prgress curves were btained in 0.2 M sdium acetate (ph 6.45) cntaining 20/Lg/ml phsphatidylserine at 25 C. 2 Ki is the dissciatin cnstant fr the initial E1 cmplex, gi* and ks/k 6 are the verall dissciatin cnstant and the rati f frward (ks) and reverse (k6) rate cnstants fr the El* cmplex (see Scheme I). stants, Ki*, fr the N-C14- t N-Cls-glucsylamines were similar ( nm), whereas the K i values, i.e., the dissciatin cnstant fr the initial EI cllisin cmplexes, were 4-25-fld greater. These results implied a direct relatinship between the ks/k 6 values and alkyl chain length (Table II). Scheme 1 depicts a mechanism cnfrming t these data which includes a slw cnfrmatinal change f EI t an El* cmplex and with the reverse cnfrmatinal change being much slwer fr the lnger alkyl-glucsylamines. Discussin / 7,5 nm nm ~< 80 2.M / / / / rime(rain) ~ // 14-GI i i i i TIME (rnin) Fig. 6. Cmparisn f the ptencies and natures f the inhibitin f human acid fl-glucsidase by N-C14-glucsylamine r -dexynjirimycin. Representative prgress curves at 20 nm f either inhibitr are shwn when enzyme and inhibitr were added simultaneusly and rapidly mixed int the substrate slutin. In preincubatin experiments (see Materials and Methds), the identical linear prgress curve fr N-C14-dexynjirimycin was btained. The inset prvided the prgress curves fr several cncentratins f N-Cx4-glucsylamine which demnstrate the slw apprach t maximal inhibitin. Assay cnditins were as in Fig. 2. The inhibitr studies reprted here prvide insight int the requirements fr the binding f sphingsine and substrate analgues t the active site f acid fl-glucsidase as well as int the ptential reactin intermediates during substrate hydrlysis by this enzyme. Previusly, the length (1-24 carbns) f the fatty acid acyl chain f glucsylceramides was shwn t have large effects n the hydrlytic rates f these substrates but small effects n their apparent affinity fr human acid fl-glucsidase [13]. On the basis f such studies and the predminantly cmpetitive nature f the inhibitin f acid fl-glucsidase by natural sphingsine, the active site was prpsed t cntain a binding site fr sphingsyl mieties whse affinity fr these cmpunds was directly related t chain length. The results btained with the sphingsine derivatives (Table I) supprt this prpsal and indicate that the active site culd ptimally accmmdate chains f carbns. The inhibitry effects f the synthetic sphingsines were rapidly reversible and they had inhibitry ptencies similar t that f natural (2R,3R)-sphingsine islated frm Gaucher disease spleens. The lack f inhibitin (up t 2 mm) f the enzyme by (1S,2S)-2-amin-1- phenyl-prpane-l,3-dil whse plar head grup cm-

8 19 psitin was similar t that f sphingsine, indicated that steric effects r inhibitr cnfiguratin als were imprtant fr determining sphingsine binding. Sarmients et al. [25] demnstrated that the 3-hydrxy erythr-n-acetyl-glucsylceramide in lipsmes was a better substrate fr acid fl-glucsidase than the crrespnding thre r ket derivatives. In the present studies a cmparable preference was nt reflected in the inhibitry ptencies f the erythr and thre sphingsines (Table I). Althugh the presence f a linear triple bnd at C4-C5 had n effect n the ptency f inhibitin, the abslute requirement fr the presence f unsaturatin [13,26] between these carbns was cnfirmed by the lack f inhibitin f acid fl-glucsidase by C20- N-acetyl-sphinganine and 4-sphinganine. This cmplete lack f inhibitin f this enzyme by the 4-sphinganine derivatives supprts the existence f a subsite within the active site with a majr preference fr 4-sphingenene mieties. The N-C10- t N-C18-glucsylamines are the mst ptent inhibitrs f human acid fl-glucsidase described and were slw-tight binding inhibitrs [22]. As such they culd prvides insight int the specificity f binding t the active site as well as the nature f the reactin intermediates during substrate hydrlysis [22]. Cmpared t the Cn-l-O-fl-glucsides [13], the replacement f the xygen at the C1 psitin with a N, i.e., the N-C,-glucsylamines, increased the inhibitry ptency by abut 106-fld. The imprtance f a N grup in prximity t C1 f glucpyranse previusly was indicated by the fld increased inhibitry ptencies f fl-glucsylamine and dexynjirimycin (1,5-didexy-5-imin-glucse) cmpared t fl-glucse [7]. Hwever, the inhibitry ptency f the N-C,-glucsylamines wuld be underestimated by abut 103 frm the additinal free energies (cmpared t glucse) f binding fr the glycn head grup and the alkyl chain: The Kiapp values fr the analgus series f N-C10 t N-C14-dexynjirimycins culd be predicted within a factr f 2-4 [13]. Als, the N-C~0- t N-Cln-glucsylamines were abut fld mre ptent inhibitrs than the crrespnding N-C~-dexynjirimycins (Table I) and the verall dissciatin cnstants, Ki*, fr the N-C10- t N-C~8-glucsylamines were similar t each ther. These findings indicate that the amin acid residues in the acid fl-glucsidase active site which interact with these N grups have high specificity fr their lcatin in the glucse ring. Tgether with previus results [7,13,25], the present findings indicate a high degree f specificity f the active site residues fr the cnfiguratin and substituent grups f C1, C2, C3 and C4 n glucpyranse. In additin t the tight binding, the presence f a N grup at the anmeric carbn f alkylglycns cnferred slw binding prperties t the N-Cx2- t N-C18-glucsylamines. Indeed, this prperty was fund nly with the N-Cn-glucsylamines and nt with the parent cmpunds, fl-glucsylamine r alkylamine, r the N-C,-dexynjirimycins. Accrding t Mrrisn and Walsh [22], the slwness, rather than the tightness per se, f binding is a prperty f transitin state (reactin intermediate) analgues during substrate reactins, since the enzyme must be frced int the prper cnfrmatinal substate t recgnize these cmpunds. The direct relatinship between the chain length f the N-C,-glucsylamines and time required t achieve maximal inhibitin was cnsistent with this cncept, since the naturally ccurring substrates fr acid fl-glucsidase have hydrphbic chains f carbns in length [27]. The finding that the slw equilibrating prperty was due t a slw cnfrmatinal change f the EI cmplex suggests that such an ismerizatin may be required fr the transitin state during substrate hydrlysis. This was supprted by the fact that the shape f the prgress curves and the tl/2 values fr develpment f maximal inhibitin were similar at ph , even thugh the ph ptimum was abut 5.8. Furthermre, changes in inizatin f active site residues cannt accunt fr these results, since catalytic activity (Vmax)cnfrmed t a diprtic [7] mdel with nly a mnprtnated active site frm being active. Cnsequently, the prgress curves mnitr the changes in hydrlytic rates f whatever amunt f mnprtnated active sites are present. In this regard, it is f interest that the N-Cn-glucsylamines did nt appear t be substrates fr acid fl-glucsidase. The dissciatin cnstant, Ki, fr the initial enzyme N-Claglucsylamine cmplex was similar t that fr the N- C14-dexynjirimycin, a rapidly reversible inhibitr f acid fl-glucsidase. This result indicates that the initial cllisin cmplexes fr bth types f N-alkyl-glycn derivatives invlve similar interactin within the active site f acid fl-glucsidase, but the presence f the N grup n the anmeric carbn (and t sme extent the chain length) was required fr (r induces) a cnfrmatinal change fr ptimal alignment f amin acid residues which may be invlved in substrate hydrlysis. The determinatin f the prtnatin state f the N grup f the N-Cn-glucsylamines shuld help further elucidate the reactin mechanism. Unfrtunately, the instability f the glucsylamines at acidic ph values precluded the needed ph variatin studies. Hwever, in phsphatidylserine cntaining buffers, the IC50 values (abut nm) btained at ph 5.5, 6.0 and 6.45 fr the N-C10- and N-C12- derivatives were nearly cnstant when crrected fr inhibitr degradatin. Furthermre, based n pk a = 5.3 [9] fr fl-glucsylamine, the unprtnated N-C,-glucsylamine wuld be in abut a 15-fld excess ver the prtnated frm at ph That is, the IC50 values, assuming exclusive binding f the prtnated inhibitr, wuld be abut 20 pm at 1.1 nm enzyme, which seems unlikely, since nly abut 2% f the enzyme wuld be bund t inhibitr. These findings

9 20 are cnsistent with either the unprtnated frms f these inhibitrs binding t the active site, r that neither prtnatin state is preferred and the crrect prtnatin is acquired within the active site. The use f these pwerful inhibitrs f acid fl-glucsidase activity tgether with site-directed mutagenesis shuld cntinue t prvide insight int the nature f active site amin acids which are imprtant fr binding and catalysis and the disruptin f these enzymatic functins in Gaucher disease [28]. Acknwledgements This research was supprted by grants t G.A.G. frm the Natinal Institutes f Health (DK36729), the Natinal Gaucher Disease Fundatin (NGF-19), the General Clinical Research Resurces branch f the Natinal Institutes f Health (RR-71), and frm Flrence and Thedre Baumritter t the Munt Sinai Center fr Jewish Genetic Diseases and t A.H.M. and D.C.L. frm the Natinal Science Fundatin (DCB ). G.A.G. is the recipient f an NIH Research Career Develpment Award (DK01351). References 1 Brady, R.O., Kanfer, J.N. and Shapir, D. (1965) J. Bil. Chem. 240, Gatt, S. and Rapprt, M.M. (1966) Bichim. Biphys. Acta 113, Desnick, R.J., Gatt, S. and Grabwsld, G.A. (eds.) (1982) Gaucher Disease: A Century f Delineatin and Research, Alan R. Liss, New Yrk. 4 Srge, J., West, C., Westwd, B. and Beutler, E. (I985) Prc. Natl. Acad. Sci. USA 82, Tsuji, S., Chudary, P.V., Martin, B.M., Winfield, S., Barranger, J.A. and Ginns, E.I. (1986) J. Bil. Chem. 261, Ericksn, J.S. and Radin, N.S. (1973) J. Lipid Res. 14, siecki-Newman, K.M., Legler, G., Grace, M.E., Dinur, T., Gatt, S., Desnick, R.J. and Grabwski, G.A. (1988) Enzyme 40, Dinur, T., Osiecki, K.M., Legler, G., Gatt, S., Desnick, R.J. and Grabwski, G.A. (1986) Prc. Natl. Acad. Sci. USA 83, Grabwski, G.A., Gatt, S., Kruse, J. and Desnick, R.J. (1984) Arch. Bichem. Biphys. 231, Berent, S.L. and Radin, N.S. (1981) Bichim. Biphys. Acta 664, Basu, A., Glew, R.H., Daniels, L.B. and Clark, L.S. (1984) J. Bil. Chem. 259, Legler, G. and Liedtke, H. (1985) Bil. Chem. Hppe-Seyler 366, Osiecld-Newman, K.M., Fabbr, D., Legler, G., Desnick, R.J. and Grabwsld, G.A. (1987) Bichim. Biphys. Acta 915, Legler, G. and Bieberich, E. (1988) Arch. Bichem. Biphys. 260, Gaver, R.C. and Sweeley, C.C. (1965) J. Am. Oil Chem. Sc. 42, Pigman, W., Cleveland, E.A., Cuch, D.H. and Cleveland, J.H. (1951) J. Am. Chem. Sc. 73, Nimbar, S., Menaldin, D., Merrill, A.H. and Litta, D.C. (1988) Tetrahedrn Lett. 29, Isbell, H.S. and Frush, H.L. (1958) J. Org. Chem. 23, Osiecki-Newman, K.M., Fabbr, D., Dinur, T., Bas, S., Gatt, S., Legler, G., Desnick, R.J. and Grabwski, G.A. (1986) Enzyme 35, Pal, R., Petri, W.A., Berenhltz, Y. and Wagner, R.R. (1983) Bichim. Biphys. Acta 729, Shulman, M., Kulshin, V.A. and Khrlin, A.Y. (1980) Anal. Bichem. 101, Mrrisn, J.F. and Walsh, C.T. (1988) in Advances in Enzymlgy and Related Areas f Mlecular Bilgy (Meister, A., ed.), Vl. 61, pp , Academic Press, San Dieg, CA. 23 Dinur, T., Grabwski, G.A., Desnick, R.J. and Gatt, S. (1984) Anal. Bichem. 136, Lwry, O.H., Rsebrugh, N.J., Farr, A.L. and Randall, R.J. (1951) J. Bil. Chem. 193, Sarmients, F., Schwartzmann, G. and Sandhff, K. (1986) Eur. J. Bichem. 160, Vaccar, A.M., Muscill, M. and Suzuki, K. (1985) Eur. J. Bichem. 146, Nilssn, O., Manssn, J.E., Hakanssn, G. and Svennerhlm, L. (1982) Bichim. Biphys. Acta 712, Grace, M.E., Graves, P.N., Smith, F.I. and Grabwski, G.A. (1990) J. Bil. Chem., in press.