Biochimica et Biophysica Acta, 544 (1978) 489--495 © Elsevier/North-Holland Biomedical Press
GLUCOSYL TRANSFERASE ACTIVITY OF BOVINE GALACTOSYL TRANSFERASE
PIET JAN ANDREE * and LAWRENCE J. BERLINER **
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210 (U.S.A.) (Received April 24th, 1978)
Summary Bovine galactosyl transferase was found to utilize UDPgiucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of giucosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPgiucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor. Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.
Introduction Galactosyl transferase (uridine diphosphate-D-galactose : D-glucose-l-galactosyltransferase, EC 18.104.22.168) of bovine milk catalyzes the two basic reactions below: UDPGal + GIcNAc Me(I!.)UDP + LacNAc
where Me(II) is restricted to certain divalent cations (e.g. Mn 2÷ or Zn 2+) but n o t * Present address: Molecular Biochemistry, Veterans A d m i n i s t r a t i o n Hospital, Kansas City, Mo. 64126,
U.S.A. ** T o w h o m rep rin t requests are to be addressed. Abbreviations: GIcNAc, N-acetYlglucosamine; UDPGal and UDPGIc, UDP-galactose and glucose, respectively; LacNAc0 N-acetyllactosamine; UDP-R, urldine 6t-diphosphate 4-(2,2,6,6-tet~'amethylplp e r i d i n y l - l - o x y ) ; 2r,3 r diaI-UDP-R, 2r,3 t dialdehydo-uridine 5P-diphosphat~4-(2,2,6,6-tetramethylp i p e r i d i n y l - l - o x y ) : the product of periodate cleavage of UDP-R; NMM, N - m e t h y l morDholine.
490 Mg2+ . In the presence of the modifier protein, a-lactalbumin, lactose synthesis occurs with glucose as the acceptor: UDPGal + glucose ~_lactalbumin Me(II) ~UDP + lactose
The donor specificity of galactosyl transferase has been examined in detail in only one earlier report where it was shown that deoxy UDPGal also functioned as a donor substrate . Furthermore, UDPGlc, the 4'-epimer of UDPGal, was shown to be an effective competitive inhibitor of UDPGal [3,4]. In past work, we demonstrated that a spin label uridyl sugar analog, UDP-R, also functioned as a competitive inhibitor for UDPGal with somewhat less potency than
UDPGIc [51. CH3 H H
O - - N ~
We have been quite interested in how the enzyme imparts its specificity towards galactose as the donor saccharide moiety and apparently not glucose, which differs only by its equatorial (rather than axial) 4'-hydroxyl group. Either the UDPGal binding site is sterically restricted at the equatorial 4' position for any group larger than a proton, or the formation of a specific hydrogen bond with an axial 4'-hydroxyl group conformationally "signals" the active site to catalyze the scission at the 1' position and subsequent transfer to the carbohydrate acceptor (glucose or GlcNAc). The present study demonstrates a marginal, yet real, glucosyl transferase activity of galactosyl transferase in the presence and absence of a-lactalbumin. Materials
UDP-R was synthesized after Berliner and Wong . UDPGal, UDPGIc, were from Sigma Chemical Company. UDPGal ([U-14C]Gal, 200mCi/mmol) and UDPGIc ([U-14C]GIc, 150--250 mCi/mmol) were obtained from New England Nuclear. Fresh raw milk was obtained from the O S U Dairy Barn. Pyruvate kinase (type 1) and a4actalbumin were from Sigma Chemical Company. Galactosyl transferase was purified by the method of Geren et al.  through the "hydrophobic" chromatography step on norleucine-Sepharose 4B, but was then followed by one or two affinity chromatography steps with a-lactalbumin,Sepharose to yield pure enzyme of specific activity, typically 16--20 units/ragat 30°C. *
phosphoenolpyruvate and N A D H
* T h e e n z y m e u n i t is c o n v e n t i o n a l l y d e f i n e d as t h e p r o d u c t i o n o f 1 / ~ m o l U D P / m i n .
Galactosyl transferase activity for reactions (1) or (2) above was measured by either the coupled spectrophotometric assay method of Fitzgerald et al.  or the radiometric assay of Trayer and Hill  at 30°C. A molecular weight of 50 000 was used as an average of the t w o catalytically identical molecular weight forms of the enzyme . Enzyme concentration was estimated spectrophotometrically using specific absorbance, Azs0nm (0.1%) = 1.61 [ 8], with a Unicam SP1800 spectrophotometer. After the procedures of Powell and Brew , two mg UDP-R were treated in 50 pl of 9 mM sodium periodate, pH 5.0, for 85 min at room temperature to produce 2',3' dial-UDP-R. Subsequently a solution containing 2.9 mM 2',3' dialUDP-R and 1.8 mg/ml galactosyl transferase in 0.03 sodium cacodylate, pH 7.4/0.17 mM MnC12, 3.1 mM GlcNAc was allowed first to equilibrate for 3 h at ambient temperature and then treated with 9 mM NaBH4 (15 rain at 0°C, 30 min at room temperature) to reduce the intermediate Schiff's base. The covalently modified enzyme after dialysis against 0.05 M Tris, pH 7.4/0.1 M (NH4hSO4/2 mM e-amino caproic acid, was assayed for residual activity and spin concentration in order to estimate the extent of labeling. ESR measurements were carried out on a Varian E-4 spectrometer (X-band) in small quartz capillaries . Results
In our NMR and ESR studies of substmte complexes of galactosyl transferase  we first noted evidence for a chemical reaction in the presumed unreactive quaternary complex Mn(II) • galactosyl transferase • UDPGIc • GlcNAc, as evidenced in the NMR by a steady increase in the water proton T1 rate, or in the ESR, by a decrease in free Mn(II) peak height. Both results were consistent with production of UDP, which forms a strong binary complex with Mn(II) in the neutral pH region. By assaying this system under similar conditions for the coupled assay for N-acetyllactosamine biosynthesis  we substituted 0.67 mM UDPGlc for the normal 0.26 mM UDPGal. The relative rate for UDP proTABLE I R F VALUES REACTIONS
Standards Galactose Glucose Lactose Cellobiose
0.70 0.80 0.38 0.48
Reactions LacNAc UDPGlc ([U14C]Gle) + GIcNAc UDPGlc ( [ U 1 4 C ] G l c ) + glucose
0 . 6 5 +- 0 . 0 3 0 . 6 9 -+ 0 . 0 4 0 . 5 0 -+ 0 . 0 3
+ 0.02 -+ 0 . 0 2 +_ 0 . 0 2 _+ 0 . 0 3
* R G I c N A c is d e f i n e d as t h e R F v a l u e r e l a t i v e t o a G I c N A c standard. S o l v e n t s y s t e m w a s b u t a n o l / p y r i d i n e / 0 . 0 1 M s o d i u m b o r a t e (2 : 2 : 1, v / v ) .
Fig. 1. R a d i o e h r o m a t o g r a m scan o f a m i x e d paper ebromatogram o f the products f r o m the L a c N A c react i o n (1) and t h a t f r o m substituting UDPGle f o r UDPGal, Conditions are d e s c r i b e d in the t e x t ,
duction in the presence of UDPGlc, GlcNAc, Mn(II) and galactosyl transferase (for the presumed transfer of glucose to GlcNAc) was 0.3% of that for N-acetyUactosamine production. This rate was negligible in the absence of GlcNAc. In order to exclude exogenous UDPGlc : galactose epimerase activity as being responsible for these results we note that in our assay mixture, if epimerase were present, UDPGal would be formed even in the absence of GlcNAc. At some time later, upon GlcNAc addition, a fast jump in absorbance reflecting a fast UDP production from the previously formed UDPGal would be expected. Such behavior was not observed, excluding this as a viable possibility. In experiments to show clear chemical proof of the proposed new reaction product, radioactive UDPGlc was substituted for UDPGal under conditions used for the N-acetyUactosamine and lactose synthesis reactions, respectively, at pH 7.4, 26°C. The reaction was run with UDPGlc ([U-14C]Glc) for 18 h, stopped by addition of Dowex RG 501-X8 mixed bed resin to remove Mn(II) and nucleotides, and chromatographed on Whatman No. 1 paper in butanol/ pyridine/0.01 sodium borate (2 : 2 : 1, v/v). * Table I summarizes R F values for these reaction products and some standards. In the absence of acceptor, no radioactive neutral products were formed. For the reaction with glucose as "acceptor", the new spot with a typical disaccharide mobility migrated with a n R F value identical to that of cellobiose, while for the reaction with GlcNAc as acceptor the new spot migrated with a n R F value in the range of, but not identical with, that for N-acetyllactosamine, as is evident from two distinct spots in Fig. 1 from a mixed chromatogram. Absolute proof of glucose incorporation in these products was shown by acid hydrolysis and rechromatography. NaBH4-reduced 2',3' dial UDP-R: A protein conjugate. A 47% active enzyme * While o t h e r s o l v e n t s y s t e m s y i e l d e d s i m i l a r r e s u l t s , t h i s s o l v e n t gave the b e s t r e s o l u t i o n o f t h e s t a n d a r d s and products under investigation.
o- c.s CH5 H diol
I~ ~ ,
UDP - R.
I I0 gou$$ Fig. 2. X-Band ESR sp ectrum of NaBH4-reduced 2',3' dlal UDP-R-galactosyl tzansferase c o m p l e x a t pH 7.4, 0.05 M Tris-HCII0.1 M (NH4)2SO412 mM e-aminocaproic acid, at 28 ± 2°C. The r o t a t i o n a l correlation time for the n i t r o x i d e m o i e t y was only a p p r o x i m a t e l y I order of m a g n i t u d e longer than that for the free label in water.
was obtained which contained 0.38 mol spin label per mol protein as measured by its spin count . Its ESR spectrum, which is shown in Fig. 2, is reflective of a highly mobile nitroxide moiety. We also noted that the presence of excess periodate inactivated native enzyme, contrary to the results of Powell and Brew . Therefore, a slightly larger fraction of inactive enzyme than that coupled with spin label was in part accounted for by this latter phenomenon, as well as that due to a small percentage of UDP-R which was hydrolyzed to diamagnetic UDP. The 2',3' dial UDP-R moiety was most certainly bound at the UDPGal site, as shown previously for the parent compound, UDP-R, based both on the 1 : 1 stoichiometry of binding and competitive inhibition behavior of the latter . Since the primary sequence is not yet available we considered amino acid analysis of the dial derivative enzyme to be no more informative than the already available results of PoweU and Brew . Discussion
From the combined NMR, ESR and catalytic activity data above it has been established that, in the presence of the carbohydrate acceptors, GlcNAc or glucose (in the presence of a-lactalbumin) bovine galactosyl transferase catalyzes the transfer of glucose from UDPGlc. The paper chromatography results have demonstrated that this acceptor was glucose in the presence of a-lactalbumin with a disaccharide product which migrated with ceUobiose. The product with GlcNAc as "acceptor" migrated in the range expected for a disaccharide~ontaining reducing end GlcNAc. While we were not able to produce suffi-
T A B L E II THERMODYNAMIC AND KINETIC BINDING EQUILIBRIA FOR URIDYLYL GALACTOSYL TRANSFERASE Derivative
K I (mM) *
K D (mM) * *
UDPGIe UDP UDPGal UDP-R
0.089 0,067 -0.38
[ 3] 
0.023 0.008 0.014 0.40
[ 14]  [ 14] [ 5]
• F o r r e a c t i o n 1, t h e c o m p e t i t i v e i n h i b i t i o n c o n s t a n t m e a s u r e d k i n e t i c a l l y . • * The equilibrium dissociation constant.
cient amounts of this latter product for a complete characterization it seems most probable that, like cellobiose, GlcGlcNAc was bonded in a ~ (1--4) linkage. In both the NMR and ESR experiments, the binary complex Mn(II) • UDP was essentially the only major product which would form under those conditions and also yield the reported effects on the Mn(II)-induced water proton relaxation rates and on the free Mn(II) ESR spectra. Since the UDPGlc concentrations used in the rate assays were well above the reported KI for this molecule [3,4] we can assume that the 0.3% relative activity reflects principally differences in keat between UDPGlc and UDPGal as substrates for this enzyme. Of course, the efficacy of UDPGIc as a competitive inhibitor towards UDPGal is understandable since the former is turned over so slowly relatively to the latter. Perhaps this reduced rate simply occurs as a result of a slight misaligment of the glucosyl phosphate ester bond with the pertinent catalytic residues. The binding of UDPGlc compares well with UDP and other derivatives, as shown in Table II. This indicates that uridylyl binding dominates in most UDP derivatives [ 5]. Thus the manifestations of reduced transferase activity with UDPGlc are probably due to improper binding of the glucosyl moiety alone. Two possible explanations for the reduced rate constant include a concomitant improper alignment of the glucosyl phosphate ester bond with the catalytic residues and/or the loss of the hydrogen bond network to the 4' axial position resulting in the loss of some conformationally linked activation mechanism (see Fig. 3). Of course, UDPGlc still acts as a competitive inhibitor towards UDPGal even though it is also a (very slow) substrate. The binding of 2',3' dial UDP-R, as evidenced by its ESR spectrum (Fig. 2) indicates that few, if any, binding interactions occur between the piperidine nitroxide and the galactosyl binding region. * The bulky size of the piperidine nitroxide (contributed in part by the flanking di-t-alkyl methyl groups) precludes a complementary snug fit to a galactosyl binding template. The high
* T h e r e a r e a p p a r e n t l y n o highly reactive n u c l e o p h f l e s i n t h i s d o n o r site as w e l l , as t h e u r i d y l a n a l o g b r o m o a c e t a m i d o - p h e n y l - u r i d y l P y r o P h o s p h a t e reacted o n l y w i t h t h i o l g r o u p s l o c a t e d e l s e w h e r e o n t h e e n z y m e ( G r u n w a l d , J . , S p e r r y , D. a n d Be~Uner, L.0 u n p u b l i s h e d r e s u l t s ) despite the p o t e n c y of this uridyl affinity label with UDP-glueose : galactose epimerase .
Fig. 3. Binding m o d e l for a galactosyl (left) or gincosyl (right) m o i e t y in the UDPGal binding site of the enzyme. The binding locus near the 4 t p y r a n o s y l p o r t i o n ma y e xa c t l y fit galactosyl m o i e t y with a potential h y d r o g e n b o n d to its axial h y d r o x y l group while a glucosyl m o i e t y is t oo crowded, and misaligns the entire UDPGIc molecule at the catalytic residues; it also lacks the 41 axial h y d r o g e n bond.
mobility of the nitroxide moiety in this covalently-bound derivative is supportive of a model with the UDPGal binding region at the protein surface which allows the nitroxide moiety free rotation. Acknowledgements This work was supported, in part, by a grant from the U.S.P.H.S. (GM 21923). P.J.A. is the recipient of an Ohio State University Postdoctoral Fellowship 1976--1977. L.J.B. is an established investigator of the American Heart Association 1975--1980. We are grateful to Dr. R.M. Mayer for helpful discussions. References 1 2 3 4 5 6 7 8 9 10 11 12 13
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