Substrate Specificity and Reaction Mechanism of Human ...

Vol. 267,No. 10, Issue of April 5, pp. 6855-6858.1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMI~ITRY 0 1992by The American Society for Biwhemistry and Molecular Biology, Inc.

Substrate Specificity and Reaction Mechanism of Human Glycoasparaginase THE N-GLYCOSIDIC LINKAGE OF VARIOUS GLYCOASPARAGINES IS CLEAVED THROUGH A ACTION M E C H ~ I SIMILAR S ~ TO L-ASPA~GINASE* (Received for publication, May 8,1991)

Vesa KaartinenS08,Tarja Mononenli11, Rein0 Laatikainenp, andIlkka Mononen+li** From the $Department of Clinical Chemistry, Kuopio University Central Hospital, the $Department of Chemistry, University of Kuopb, and the BCenter for Diagnostic Biotechnology,A. I. Virtanen Institute, SF-70210Kuopio, Finland and the \]Divisionof Medical Genetics, Children's Hospital of Los Angeles, Los Angeles, California 90054-0700

Human glycoasparaginase (N4-(@-N-acetyl-~-glu-19-kDa light subunits and 25-kDaheavy subunits. The Lcosaminy1)-L-asparaginase,EC 3.5.1.26) hydrolyzes a asparagine analogue 5-diazo-4-oxo-L-norvaline (DONV)' inseries of compounds that contain L-asparagine residue hibits its activity rapidly and irreversibly. This is due to the with free a-amino and a-carboxylgroups. Substrates chemical modification of the amino-terminal end of the light include high mannose and complex type glycoaspara- subunit located near or inthe active site of glycoasparaginase gines as well as those that lack the di-N-acetylchito- (2). biose moiety, L-aspartic acid @-methylester and LHuman leukocyte glycoasparaginase has many properties aspartic acid 8-hydroxamate. The enzyme is inactive in common with bacterial and plant L-asparaginases: (i) it toward L-asparagine and L-glutamine and glycoaspar- has a polymeric subunit structure (2,4). (ii) There is a marked agines containing substituted a-amino and/or a-carsequence homology between the light chain of human gIyboxyl groups. In the presence of the acyl acceptor hydroxylamine, glycoasparaginase catalyzes the syn- coasparaginase and bacterial and plant L-asparaginases (2). thesis of L-aspartic acid 8-hydroxamate fromaspartyl- (iii) Glycoasparaginase and L-asparaginases are inhibited by glucosamine, L-aspartic acid @-methylester, and L- DONV (2, 5). Studies on the reactions of Escherichia coli Laspartic acid. "C NMR studies using 180-labeled L- asparaginase with an acyl acceptor, hydroxylamine, along aspartic acid demonstratethat glycoasparaginase cat- with oxygen exchange reactions between water and L-aspartic acid have suggested a mechanism that involves an acyl enzyme alyzes an oxygen exchange between water and the carboxyl groupat C-4 of L-aspartic acid. These results intermediate (6). The mechanism of the reaction catalyzed by indicate that glycoasparaginase reacts as an exo-hy- glycoasparaginase has not been reported. Genetic deficiency of glycoasparaginase in humans causes drolase toward the L-asparagine moiety of the substrates and the free a-amino and a-carboxyl groupsare aspartylglycosaminuria (McKusick 20840). Aspartylglucosarequired for theenzyme reaction, The resultsare con- mine is the major metabolite accumulating in tissues and sistent with an L-asparaginase-like reaction pathway body fluids in the disease (7), but urine of aspartylglycosawhich involves a @-aspartyl enzyme intermediate. minuria patients also contains several other glycoconjugates Since glycoasparaginase is active toward a series of with L-asparagine at the reducing end (8-10). Some of these structurally different glycoasparagines, we suggest the compounds can be structurally derived from known N-glycorevised systematicname of N4-(@-glycosyl)-~-asparag- sidic carbohydrate chains (7), whereas some of the cominase for the enzyme. pounds, like a galactosyl derivative of aspartylglucosamine (GalGlcNAc-Asn) and itssialylated forms that lack the di-Nacetylchitobiose moiety (lo), are found in aspartylglycosamiGlycoasparaginase is a lysosomal enzyme that catalyzes the nuria. Experimental studieswith model compounds on human glycoasparaginase are necessary for better understanding of following reaction (1). the biochemical changes in aspartylglycosaminuria and the iV"(P-N-acetyl-D-glucosaminyl)-L-asparagine (aspartylglucosamine) catabolism of glycoasparagines in general. paper reportsthe findings on the enzymatic properties + H 2 0 + L-aspartic acid + 1-amino-N-acetylglucosamine of This human glycoasparaginase. The reaction mechanism was 1-Amino-N-acetylglucosamineisfurther cleaved nonenzy- studied by kinetic analysis using the acyl acceptor hydroxylmatically to form ammonia and N-acetylglucosamine (1). The amine. The activity of glycoasparaginase toward various glyhuman leukocyte glycoasparaginasehas recently been purified coasparagines and L-asparagine analogues was measured. and characterized (2), its cDNA cloned and the nucleotide Based on our resultswe propose that glycoasparaginasereacts sequence determined (3). The enzyme is a tetrameric glyco- with various glycoasparagines as an exo-type hydrolase, and protein with an apparent molecular mass of 88 kDa containing ~

* This work was financially supported by the Academy of Finland. The costs of publication of this article were defrayed in part by the payment of page charges. This articlemusttherefore be hereby n taccordance " with 18 U.S.C. Section 1734 marked ' ~ ~ v e r t ~ e m ein solely to indicate this fact. +*To whom correspondence should be addressed Dept. of Clinical Chemistry, Kuopio University Hospital, SF-70210 Kuopio, Finland. Fax: 358-71-173179. Tel.: 358-71-173180;

The abbreviations used are: DONV, 5-diazo-4-oxo-~-no~aline; CrnCys, ~-Carbox~ethyl-L-cysteine; GalGlcNAc-Asn, @-N-acetylla~tosamine-L-asparagine; HPLC, h i g h - ~ r f o r ~ a n cliquid e chromatography; Man~GlcNAc~-Asn, high mannose type glycoaspatagine; NeuAc*Gal~Gl~NAc~Man~-Asn, bi-antennary glycoasparagine; NeuAca~Z-3)GAlGlcNAc-Asn,~-neuraminyl-a2-3-N-acetyI~ac~samine-L-asparagine; NeuAca(2-6) GAfGlcNAc-Asn, 8-neuraminyl~2-6-N-acetyllactosamine-~-asparagine; PTC, phenylthiocarbamy~ TSP, 3-trimethylsilyl-1-propane sulfonic acid.

6856

Reaction Mechanism of Glycoasparaginase TABLE I K , ualues and relative rates of hydroLysis for various

aspartic acid. The initial rate of the reaction 53 pM/min (3.3 units/mg of protein), aK, of 0.7mM and a kcatof 309X min". glycoasparaginase substrates The accumulation of L-aspartic acid 8-hydroxamate is linear Romannumerals given in brackets refer to the corresponding with time only in the beginning of the reaction, because the schematic structures shown in Fig. 3. Relative rates have been calculated from VmaXvalues of hydrolysis of various substrates. The enzyme hydrolyzes the compound and hydroxamic acid coefficient of variation for Vmaxand K,,, values for hydrolysis of formed as a product is an effective competitive inhibitor (Ki aspartylglucosaminewere 20 ( n = 3) and 11% ( n = 6), respectively. = 5 pM) of the hydrolysis of aspartylglucosamine by glycoasNM, not measured. paraginase. It is thus apparent that glycoasparaginase is capable of catalyzing the hydroxylaminolysis and hydrolysis of Compound Relative rate K, aspartylglucosamine at similar rates. Formation of L-aspartic % m&f acid P-hydroxamate with L-aspartic acid @-methylester at pH @-N-Acetylglucosamine-L-asparagine(I) 0.11 100 7.5 had a slightly slower initial rate (30 pM/min; 1.9 units/mg @-N-Acetyllactosamine-L-asparagine(11) 88 0.15 @-N-Acetylneuraminyl-n2-3-N-acetyllacto0.16 71 of protein) than that observed with aspartylglucosamine. Forsamine-L-asparagine(111) mation of L-aspartic acid b-hydroxamate with L-aspartic acid ~-N-Acetylneuraminyl-n2-6-N-acetyllacto53 0.16 occurred with an initial rate of 0.14pM/min (9 milliunits/mg samine-L-asparagine(IV) of protein). The initial rate of the hydroxylaminolysis at pH 85 NM High mannose type glycoasparagine (V) 7.5 in 5 mM aspartylglucosamine had a linear correlation to 65 NM Bi-antennary glycoasparagine (VI) a-Methyl ester-@-N-acetylglucosamine-L0 the glycoasparaginase concentration,and no reaction was asparagine observed in the absence of the enzyme. 0 a-N-Acetyl-P-N-acetylglucosamine-L-asparKinetics of the glycoasparaginase catalyzed exchange of "0 agine between water and the C-4 8-carboxyl group in L-aspartic 0 a-N-PTC-P-N-acetylglucosamine-L-asparaacid was studied by 13CNMR (Fig. 1).In the beginning of the gine exchange reaction, the concentration of ~-[4-'~C,'~0,'~0]as0.16 63 @-L-Aspartylmethyl ester @-L-Aspartylhydroxamate 4 0.94 partic acid increased, whereas upon prolonged incubation the L-Asparagine 0 amount of both L - [ ~ - ' ~ C , ' ~ Oand ~ ] -~-[4-'~C,'~O,'~O]aspartic L-Glutamine 0 acid decreased (Fig. 1).A kinetic analysis of quantitative data for the exchange gave a pseudo-first-order rate constant (1.2 s-'. Glycoasparaginase catalyzed the oxygen its reaction mechanism involves a @-aspartylenzyme inter- f 0.2) X exchange reaction between water and L-aspartic acid with a mediate. specific activity of 17 milliunits/mg of protein, which is of the MATERIALS AND METHODS~ same order of magnitude as the rate of formation of the Laspartic acid 8-hydroxamate from L-aspartic acid (9 milliunRESULTS AND DISCUSSION its/mg of protein). This suggests that L-aspartic acid and A series of glycoasparagines was tested as potential sub- water associate with glycoasparaginase independently of hystrates for glycoasparaginase. The relative hydrolysis rates of droxylamine. These results are consistent with a mechanism glycoasparagineswith larger N-glycan structures were slightly of action, which involves a @-aspartylglycoasparaginase inlower and the Michaelis constants higher than those toward termediate. A plausible mechanism for hydrolysis of the N-glycosidic aspartylglucosamine (Table I). Glycoasparagines lacking the di-N-acetylchitobiose moiety and typically accumulating in linkage i s shown in Fig. 2. We propose that glycoasparaginase body fluids and tissues of aspartylglycosaminuria patients catalyzes three symmetrical reactions (Fig. 2 A ) in thesimilar were hydrolyzedwith rates comparable with those having the way as described for L-asparaginase I1 of E. coli (6). The 1chitobiose structure (Table I). Glycoasparaginase was able to amino group in the liberated glycan moiety is further nonencatalyze the breakdown of L-aspartic acid @-methylester with zymatically cleaved to form ammonia and an oligosaccharide a hydrolysis rate that was of the same order asthat of with N-acetylglucosamine at the reducing end as shown by glycoasparagines with the N-glycan moiety composed of two Makino et al. (1) (Fig. 2B). Based on the substrate specificity or more monosaccharides (Table I). At pH 7.5, glycoaspara- and themechanism of action of glycoasparaginase,we suggest ginase catalyzed the hydrolysis of L-aspartic acid p-hydroxa- the revised systematic name N4-(@-glycosyl)-~-asparaginase mate at a maximal rate of about 4% of that of the hydrolysis for the enzyme instead of the current name N4-(P-N-acetylof aspartylglucosamine. These data strongly suggest that glycoasparaginase reacts as an exo-hydrolase toward the L-asparagine moiety of the substrates. Although L-aspartic acid 8-methyl ester is agood substrate for both glycoasparaginase and E. coli L-asparaginase, these two asparaginases show profound functional differences: glycoasparaginase was completely inactive toward L-asparagine while E. coli L-asparaginase was completely inactive toward the glycoasparagines (data not shown). In the presence of hydroxylamine, glycoasparaginase cata0 1000 2000 3000 lyzed the formation of L-aspartic acid p-hydroxamate with Time (min) aspartylglucosamine, L-aspartic acid &methyl ester, and LFIG. 1. Kinetics of "0 exchange between ~ - [ 4 - ' ~ C , ' ~ O ~ ] a s Portions of this paper (including "Materials and Methods," part partic acid and Ha'60 at 37 OC. The exchange reaction was folof "Results and Discussion," and Figs. 3 and 4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

lowed by recording 13C NMR spectra at the C-4 region of aspartic acid at various time intervals. The concentrations (mM) of ~ - [ 4 acid (O),and L'3C,'802]asparticacid (W), ~-[4-'~C,'~O,'~O]aspartic [4-'3C,'602]asparticacid (0)are plotted versus incubation time. A pseudo-first-orderrate constant of (1.2k 0.2)X s-' was obtained.

Mechanism Reaction 1 GlvcoNb

(A) GlymAsn

3

11

.

&

fNH20H

REFERENCES

E M

(8) G W H

+ H20

+Giycm

6857

Acknowledgments-We thank Dr. Erkki Kolehmainen, Ph.D., Department of Chemistry, University of Jyvaskyla, Finland, for his assistance in the 'H NMR analysis of glycoasparagines and Dr. John Tomich, Ph. D., Division of Medical Genetics, Childrens Hospital of Los Angeles, for valuable comments on the manuscript.

w

# .-&E-. 2

of Glycoasparaginase

+ NH3

FIG. 2. A scheme for hydrolysis of the N-glycosidic linkage. A , the reactions catalyzed by glycoasparaginase. GlycoNH,, carbohyE-Asp, @-aspartyl drate moiety with 1-amino-N-acetylglucosamine; enzyme intermediate; @-AHA,L-aspartic acid @-hydroxamate.B , nonenzymatic formation of ammonia and an oligosaccharide with N acetylglucosamine at thereducing end according to Makino et al. (1).

D-glucosaminy1)-L-asparaginasein order to betterdescribe its properties. of human kidney hyThe endo-P-N-acetylglucosaminidase drolyzes efficiently the di-N-acetylchitobiose moiety of high mannose type glycopeptides buthaslittle activity toward desialylated complex type glycoasparagines (19). If this would apply to endo-B-N-acetylglucosaminidases in other tissues as well, it would suggestthat degradation of the N-linkage region of complex type glycoasparagines would preferentially occur through successive action of proteolytic enzymes, L-fucosidase, glycoasparaginase, and chitobiase (20). The present study clearly demonstrates that glycoasparaginase effectively hydrolyzes complex type glycoasparagines. Theoretically, the presence of both of these pathways in vivo would give the best explanation so far for the structures of accumulating glycoasparagines in aspartylglycosaminuria. Aspartylglucosamine would thus mostly originate from the action of endo-P-Nacetylglucosaminidase on high mannose type glycoasparagines. A series of glycoasparagines with structurally different N-glycans derived from known complex-type carbohydrate chains could beexplained by action of exoglycosidases on the glycoasparagines accumulating in tissues. This, however, remains to be shown, since little experimental data is available about the properties of human endo-@-N-acetylglucosaminidase and chitobiase.

1. Makino, K., Kojima, T., and Yamashina, I. (1966) Biochem. Biophys. Res. Commun. 24,961-966 2. Kaartinen, V., Williams, J. C., Tomich, J., Yates, J. R., III., Hood, L. E., and Mononen, I. (1991) J. Bwl. Chem. 266,5860-5869 3. Mononen, I., Heisterkamp, N., Kaartinen, V., Williams, J. C., Yates, J . R., 111, Griffin, P., Hood, L. E., and Groffen, J. (1991) Proc. Natl. Acad. Sci. U.S. A. 88, 2941-2945 4. Wriston, J. C. (1971) Enzymes 4, 101-121 5. Peterson, R. G., Richards, F.F., and Handschumacher, R. E. (1977) J. Biol. Chem. 252,2072-2076 6. Ehrman, M., Cedar, H., and Schwartz, J. H. (1971) J. Biol. Chem. 246,88-94 7. Beaudet, A. L.,and Thomas, G. H. (1989) in The Metabolic Basis ofznherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) pp. 1603-1622, 6th Ed., McGraw-Hill, New York 8. Pollitt, R. J., and Pretty, K., M. (1974) Biochem. J. 141, 141146 9. Lundblad, A., Masson, P. K., Norden, N. E., Svensson, S., Ockerman P.-A., and Palo, J. (1976) Eur. J. Biochem. 67,209-214 10. Sugahara, K., Funakoshi, S., Funakoshi, I., Aula, P., and Yamashina, I. (1975) J. Biochem. (Tokyo) 78,673-678 11. Hakomori, S. (1964) J. Biochem. (Tokyo)55,205-208 12. Stellner, K., Saito,H., and Hakomori, S.-I. (1973)Arch. Biochem. Biophys. 155,467-472 13. Mononen, I. (1982) Carbohydr. Res. 1 0 4 , 1-9 14. Vliegenthart, J. F., Dorland, L., and Van Halbeek, H. (1983)Adu. Carbohydr. Chem. Biochem. 41, 209-374 15. Kaartinen, V., and Mononen, I. (1990) Anal. Biochem. 190,98101 16. Tarentino, A. L., and Maley, F. (1969) Arch. Biochem. Bwphys. 130,295-303 17. Lipmann, F., and Tuttle, L. C. (1945) J. Biol. Chem. 159, 21-28 18. Rohm, K. H., and Van Etten, R.L. (1986) Arch. Biochem. Biophys. 244, 128-136 19. DeGasperi, R., Li, Y.-T., and Li, S.-C. (1989)J. Biol. Chem. 2 6 4 , 9329-9334 20. Aronson, N. N., and Kuranda, M. J. (1989) FASEB J. 3,26152622

SUPPLEMENTAL MATERIAL TO S U B S T R A STPEE C I F I C I T AY N RD EACTIO MNE C H A N I SH OM U F MAN OF VARIOUS G L Y C O A S P A R A G I N ATSNH E. .E GLYCOSID LI C NKAGE CLYCOASPARACINES IS CLEAVEDTHROUGHAREACTIONMECHANISMSIMILAR T O L-ASPARAGINASE BY VESA KAARTINEN, TARJA MONONEN, REIN0 LAATIKAINEN AND lLKKA MONONEN

Materials and propane-sulphonic Methods and(TSP) acid

--

Glycoasparaginarc was purified to homogeneity from human leukocytes as previouslydescribed(2).Thepurity of theenzymepreparation was estimated to be aver 90 % on native gel clccsophorcris, and it had a specificactivity of 3.8 Ulmg protein for the hydrolysis of asparrylglucosaminc. Arpartylglucosamine (0-N-acetylglucoramineester. L-aspartic acid 0-hydroramate and asparaginc), L-aspartic acid &-methyl wccc from Sigma Chemical Company. St. Louis phenylisothiocyanate (sequencing grade) MO., U.S.A. GalOl-4GlcNAc-Am,MangGlcNAc2-Asn. and N e u A c ~ G a l 2 G l c N A c 4 M a n 3 - A s n were products of BioCarb. Lund. Sweden. NeuAcaZ-3GalUI-4GlcNAc-Arn and NeuAca2from urine of aspartylglycosaminuria patients (LO). 6GalB1-4GlcNAc-Am were purified We ascertained their S ~ ~ U C ~ U T Cby S analysis of the componentmonosaccharides as their partially methylated alditol acetates by gas liquid chromatography-mass spectrometry (11-13)andcompleteglycoeonjugates by 280MHz I H NMR spectroscopy(14). a-Methyl ester. a-N-acetyl and a-PTC-derivatives of aspartylglucosamine were prepared as grade) w a s from Pierce Chemical described (2). Triethylamine(sequencing Company Rockford. 1L. U.S.A. A kit for protein assay was from Bio-Rad Laboratories.Richmond,CA: U.S.A. Glutamate oxalacetate transaminase. malate dehydrogenase. a-ketoglutarate and 0nicotinamide adenine dinucleotide, reduced form, were purchased from Bochringer Mannheim GmbH. Mannheim. F.R.G. Hydroxylamine hydrochloride. 3-(trimcthylsilyl)-l-

H z 1 8 0 were products of Chemical Company, Aldrlch. Steinheim. F.R.G. DL-[4-13CIaspartic acid was purchased from MSDIsotopcs.Divisionof Merck Frosat Canada Inc.. Montrcal. Canada.

- Glyeoarparaginasc hydrolase activity toward different glycoasparagines was assayed by measurmg the L-aspartic acid amount liberated with by high-performanceliquidchromatography(15). respect to theinternalstandard,CmCys, Todetermine the initial velocities. we took aliquats from the reaction mixture after an incubation of 5 . 15 and 30 minuter. The assay conditions were selected so that the reaction was proportional to time a t least for 15 minutes. We studied the action of glycoasparaginase on a-N-acetylated or a - c a r b o x y m c t h y l a t c d aspartylglucosamine derivatives as descfited (2). One unit (U) of enzyme caused loss of I pmol of substrate per We canicd out the kinetic studies with the purified minute under standard conditions. to Tarentino and Maley(16). cnzyme by a Continuous spectraphotomclricassayaccording Least rquarc Lineweaver-Buckanalysis was used in thedetermination of K m . Vmax and kcat(kcat=Vmaxlcnzymeconcentration). acid 0-hydroramate c acld 0 h v - Formation of L-aspartic during the hydrolysis of the substrates was measured according to Lipmann and Tuttle (17). The incubation mixture (125 pl) contained 0.4 M hydroxylamine. 0.5-2.0

pg

Reaction Mechanism of Glycoasparaginase

6858 glycoarparaginase (3.8 Ulmg protein) and 0

.5

m M substate in 50 m M Tris-HCI huffcr, p H

1.5. -3 1

- We obtained DL-[4-13C.I8021 aspartic acid from DL-14-13CI-

aspartic acid as described (18). The pu"ty of the I8Og-laheled aspartic acid was over 95 % according to HPLC analysis (15). The chemical shifts of the U-carboxyl carbon (C-4) of aspartic acid and their integrated peak areas indicated that the preparation was a mixture containing 12 % of DL-[4-13C,18021- and 28 % of DL-[4-13C.180.1601 aspartic acid. We studied the oxygen exchange reaction i n the presence and absence of glycoasparaginase using I 3 C N M R spectroscopy. The assay mixture contained 8 wmol of I8O-laheledDL-1413CI aspartic acid, 160 pg of glycoasparaginase (3.8 Ulmg protcin). 3 mg of TSP. 303 P I of D 2 0 and 5W p l of 50 m M phosphate buffcr. pH 1.5. Thc sample was pipetted to a 5 mm NMR tubeand a I 3 C N M R spectrum was recorded with a Brvker A M 400 Wb-spectrometer opc:&fing at 1 0 0 MHz, using a 5 m... iiivirse probe at 310 K. Spectrh -iie recorded at various limeintervals and accumulated with a total of 80 scans using composite pulse dccoupling. 1 kHz sweep width and 16 E acquisition time. A l l chemical Shifts w i l l he prercnted witha reference to TSP. In kinetic experiments. the amount of L-14-13C.18021and L-14-13C.180, 1601 aspartic acid in the incubationmixture at various time points was calculated assuming that 50% of those compounds i n the origmal preparations were their corresponding D-isomers.

Results and Discussion

exchance of I 8 0 bctween wafer and L-asoartic a c i d - The formation of L-aspartic acid I-hydroxamate from L-aspartic actd indtcater that glycoasparaginase activates the &carboxyl group (C-4) of L-aspartic acid as the L arparaginases (6). To obtain further evidence for this. we studied an oxygen transfer between 18O-labeled aspartic acid and water by I 3 CN M R . The chemical shift of the 8carboxyl carbon (C-4) of L-aspartic acid i s about 180 ppm i n respect to TSP. Incubation of D L - [ 4 - 1 3 C , 1 8 0 2 1 aspartic acid with human leukocyte glycoarparaginase in phosphate buffer. rcsullrd i n a gradual growth of the L.[4-13C.16021-signaI at 0.053 ppm downfield 4). This indicates I80 exchange hetween water and the C-48-carboxyl group in L-aspartic acid. In the beginning of the reaction. an increase of the DL-[4-13C,~60,~80]-~ignal at 0.0266 ppmdownfield from the DL-14-13C.18021signal was observed. whereas upon prolongedincubation the intensities of both the DL-1413C.1802]- andthe DL-[4-13C,160,180]-,ignal decreased (Fig. 2). After an incuhatmn of

from the DL-[4-13C,18021-,ignal(Fig.

48 h, the signal of ~-14-13C.18021 aspartic acid had practically disappeared. Since the1413CI-asparticacid preparation used i n this study was an equimolarmixture of L- and D isomers. the reaction stopped when 50 40 of 1 8 0 had been replaced by 160. The enzyme retained 90 40 of itsactivityduring the NMR experiment: no oxygen exchange reaction was detected i n the absence of glycoarparaginase.

B

Hyjrolvsis of &.wasgwazines and L-awaraeine a n a l o e m - We tested a series of glycoasparagincs (Fig. 3) as potential rubstratcs far human leukocyte glycoarparaginase. The enzyme hydrolyzed a11 of those. i n which thc L-asparagine moiety had both a frcc a amino and a-carboxyl group (Table I). Glycoasparaginasc had a K m of 0.11 mM and kcat of 333 x min-1toward aspartylglucosamine. I t hydrolyzed glycoasparagincs containing N acetylneuraminic acid i n the glycan moiety (compounds 111 and IV) with a rate slower than the corresponding asialo compound 11. The hydrolysis ratc of the N-glycosidic linkage of the high mannosc type glycoasparagine war 1.3-fold higher than that of hiantennary glycoasparagine (Table I). Glycoasparaginasc was completelyinactivetoward Lasparagine and L-glutamine (Table I).

Compound

[I-.

"

I II

Ill, I V

VI Fig 3. Schematic structure of & s o a s w i n e s

i

[I-.-. [I-.-.-* [I-.-.-O

/O-.-@-+ '0-.-.-*

used i n this studE

The location of the N-glycosidiclinkagehydrolyzedby glycoasparaginase i s indicated by L-Asparagine ( 0 ); N-acctyl-0-D-glucosamine ( 0):D-mannose ( 0 ); B-Dgalactose ( e ); N-acetyl-a-D-neuraminic acid (

an arrow.

+).

Fig. 4. 1 8 0 ~ x c h a n . e ~ D" L

-

1

4

-

1

3

C

1

8

&

~

2

1

6

Examples of the 13CNMR spectra at the C-4 region at diffcrenl time points. The reaction conditions were as indicated under "Materials and Methods".

~

o

~