Eur. J. Biochem. 83, 179-188 (1978)

Bovine Galactosyl Transferase Substrate . Manganese Complexes and the Role of Manganese Ions in the Mechanism Alexander D. TSOPANAKIS and David G. HERRIES Department of Biochemistry, University of Leeds (Received September 12, 1977)

The role of Mn2+ in the reaction catalysed by the galactosyl transferase of bovine milk and colostrum was studied by steady-state kinetic methods at pH 7.4 and 37 "C. The association constants for the binding of Mn2+ by UDP-galactose and by the buffer species used (3-[N-morpholino]propanesulphonate) were measured by electron spin resonance spectroscopy and the concentrations of all the species present in the substrate mixture of Mn2+, UDP-galactose and N-acetylglucosamine were calculated. Previous evidence indicated that high Mn2 concentrations activate the reaction beyond what is expected from the Michaelis-Menten equation. At such levels (up to 40 mM), the concentration of free Mn2+ was found to be proportional to total Mn within 3%. Experimental data produced as a function of total Mn concentration and total UDP-galactose (i.e. free UDP-galactose plus MnUDP-galactose) concentration could be interpreted in terms of rate equations written in relatively simple form with free Mn2+ and total UDP-galactose concentrations as variables, corresponding to a large variety of kinetic models involving Mn2+,UDP-galactose and MnUDP-galactose as substrates. A group of related mechanisms could not be eliminated at this stage and experiments were carried out in which the concentrations of two of the substrates were varied at either a constant ratio or a constant product depending on how they were involved in the Mn2+: UDP-galactose association reaction. A choice between the two final mechanisms was made by applying statistical non-linear least-squares methods. The most likely mechanism was one in which, after the addition of Mn2+ to the enzyme, the substrates UDP-galactose and MnUDPgalactose added as alternatives to produce two branches, in each of which was added N-acetylglucosamine followed by the release of N-acetyllactosamine, then the Mn2 complex of UDP, and finally (in the branch in which MnUDP-galactose had been the substrate) free Mn2+. +

+

The transfer of galactose from UDP-galactose to N-acetylglucosamine is catalysed by the enzyme galactosyl transferase [l]. Together with cl-lactalbumin in the lactating mammary gland, this protein forms the lactose synthase system [2] in which glucose functions as the galactosyl group acceptor. The enzyme obtained from bovine milk or colostrum has at least two metal ion binding sites, one of which is absolutely specific for Mn2+ [3]. At pH 7.4 and 37 "C in Mops bulTer, Mn2+concentrations up to 50 mM increase the initial Abbreuiutiuns. GlcNAc, N-acetylglucosamine; LacNAc, N acetyllactosamine; Mops, 3-(N-morpholino)propanesulphonicacid ; MnUDP and Mn,UDP, the 1 : 1 and 2: 1 complexes of Mn2+ with UDP; MnUDP-Gal or MnUDP-galactose, the MnZt complex with UDP-galactose; UDP-Gal, UDP-galactose. Enzymes. Lactose synthase (EC 2.4.1.22); galactosyl transferase or N-acetyllactosamine synthase (EC 2.4.1.38).

velocity for the human milk enzyme to an extent greater than expected on the basis of the MichaelisMenten relationship [4]. An analogous result has been found for the bovine colostrum enzyme [5]. The present work was undertaken to investigate the possibility that these effects are due to the Mn2+ complex with UDP-galactose being a more effective substrate than the free nucleotide-sugar, and thus being responsible for the apparent activation by Mn2 . The release of the complex MnUDP as a product has already been postulated [4]. It appears from the results reported here that Mn2+ is always the first ligand to react with the enzyme, followed by an alternative addition of UDP-galactose and MnUDP-galactose. From either of these substrates, the nucleotide is released as MnUDP, the extra Mn2 in the MnUDPgalactose branch appearing last as the free ion. +

+

180

Role of Manganese Ions in Galactosyl Transferase

MATERIALS AND METHODS Cyanogen bromide was obtained from Koch-Light Laboratories Ltd, scintillation grade 2,5-diphenyloxazole and naphthalene from Fisons Scientific Apparatus Ltd and nucleotides, nucleotide-sugars, GlcNAc and Tris from Sigma London Chemical Co. Ltd. UDP['4C]galactose was obtained from the Radiochemical Centre, hmersham, and New England Nuclear Corporation, Sephadex gels and Sepharose 4B from Pharmacia (G.B.), and all other reagents from BDH Chemicals Ltd. either AnalaR or the best available grade. Mops was recrystallised from ethanol/water ( l o p , viv). a-Lactalbumin was prepared from unpasteurised goat milk according to the method of AschaRenburg and Drewry 161 with modifications as suggested by Fox et ul, [ 7 ] . The protein was finally purified by Sephadex G- 1 00 and DEAE-cellulose chromatography. The Sepharose 4B derivative was made by direct coupling after activation of the Sepharose with cyanogen bromide at alkaline pH [8]. UDP-Sepharose was prepared from UDP-hexanolamine synthesised by the imidazolide method and coupled with Sepharose 4B according to Barker et al. ~91. Bocine Milk Galuc.to.iyl Transferase Ilnpasteurised bovine skim milk obtained from a local dairy was processed as described by Khatra et ul. [4] for the human-milk enzyme, with the replacement of MnCl, by MgCl, . The purified enzyme was stored frozen in aliquots of 1 ml at -20 'C, with an added bovine serum albumin concentration of 1 mg ml-'.

Bo 2:in e Co10s t i

w i

Gulac tosy 1 Transjerase

The procedure of Barker e f al. [9] was applied to bovine colostrum from the University of Leeds Field Station. After affinity chromatography on UDPSepharose and x-lactalbumin-Sepharose, the purified enzyme was stored in the same way as the milk enzyme. Enzj nip ('~ilui.cicteri,sution

Polyacrylamide gel electrophoresis in 10% acrylamide gels in the presence of sodium dodecyl sulphate was carried out a5 described by Weber and Osborn [lo]. Molecular weights were estimated by comparing the mobilities with those of standard proteins.

The acid dissociation constant and the stability constant for Mn2+ binding were evaluated at several points on the titration curves, with the assumption that H + and Mn2+ compete for the morpholine ring nitrogen atom on a 1 : l basis. Whcn MnCI, was present, pH values above about 8.5 were excluded from consideration because of incipient precipitation, Titration of Mn2 ' with LigandA by Electron Spin Resonunee Speetroheopy

The ESR spectra of solutions of 1 mM MnC1, in various concentrations of ligand were measured at 18 "C and pH 7.4 using a Decca X-1 9-GHz spectrometer with a 9-in (23-cm) magnet (Varian Associates), Some experiments were also carried out at 37 C to estimate the effect of temperature on the association constants. The results were analyscd by assuming that the height of each peak in the first derivative spectrum is proportional to the concentration of uncomplexed Mn2 with the bound form contributing negligibly to the spectrum (p. 26 of [12]). The data were then fitted by Eqn (11, nM p=(1 1 K-'+M +

where is the ratio of bound Mn2+ concentration to total ligand concentration, M is the concentration of free Mn2+, n is the number of Mn2 ions bound per mole of ligand, with the assumption of identical and independent sites, and K is the association constant in terms of sites. The calculations were carried out by a computer programme that used the algorithm described by Cleland [13] for fitting the MichaelisMenten equation to which Eqn (1) is analogous. UDP-glucose was used in place of UDP-galactose and the association constants assumed to be the same. Except in the case of UDP, the final values of K were determined by fitting a modified form of Eqn ( 1 ) in which n= 1. Calculation qf Individual Concentrations in the Metal- Ligand System For a system in which several ligands L, bind with association constants K, to a single metal ion M, the relationship between [MI and the total metal concentration M , is given by Eqn (2) in which L,, is the total concentration of the ith ligand and the subscripts i identify the ligands and do not indicate stoichiometry (which is assumed to be 1 :l),

Potentiometrit Titrution o j Mop3 in the Presence und Abwnw of MnC'I, Mops waa titrated with NaOH using a glass electrode in the presence and absence of an equimolar amount of MnCI, as suggested by Good et al. [ l l ] .

This equation was solved for [MI by applying the Newton-Raphson iterative method with the initial estimate of [MI set at M,.

A . D. Tsopanakis and D. G. Herries

181

It should be noted that the association constants appearing in Eqns (1) and (2) are pH-dependent quantities.

(4)

K is the association constant defined in Eqn ( 5 )

Kinetic Studies Galactosyl transferase activity was measured as described previously [I41 in 50 mM Mops buffer pH 7.4 and 37 "C. Stock solutions of UDP-['4C]galactose, 6.4 mM, were prepared containing 550 or 640 counts x min-' x nmol-'. Velocities were expressed as nanomoles of product formed per litre per second (i.e. nM s-'). In the standard assay for enzyme activity, substrate concentrations were fixed as follows : MnCI, , 10 mM ; GlcNAc, 20 mM; UDP-Gal, 0.64 mM; and enzyme, 20 pl, in a total reaction volume of 100 pl. The bovine serum albumin concentration was 0.2 mg ml-', and the enzyme concentration between 0.04 and 0.07 mg ml-'. In calculating the individual concentrations of the components of the Mn2+-UDP-Galsystem, allowance was made for complex formation between Mn2+ and the anion of Mops. The association constants used were those reported in this paper. Initial velocity/concentration data were analysed by fitting the appropriate rate equation by statistical methods. A choice among rate equations was made on the basis of the lowest experimental variance, i.e. the sum of squares of the differences between each experimental velocity and its calculated value, divided by the difference between the number of observations and the number of parameters being estimated [14]. Computer programmes based on the method of Cleland [13] were used in simple cases, but in general the more effective algorithm of Marquardt [15] proved satisfactory. This was available to us as Nottingham Algorithms Group Library programme E04GAA through the University of Leeds Computing Service. Rate equations were derived by application of either the method of King and Altman [16],or for complicated cases a computer programme for the expansion of determinants based on the report of Silvestri and Zahner [17]. Because of the high Mn2+ concentrations being investigated, and the relatively small complexing with UDP-galactose and Mops, the calculated concentrations of free Mn2+ were found to be proportional to the total Mn2+ concentration to better than 3%. The rate equations in terms of the concentrations of the species Mn2 , UDP-galactose and MnUDP-galactose (denoted respectively by M , A and C) could thus be simplified by being rewritten in terms of A4 and total UDP-galactose (Ao),after applying Eqns (3) and ( 4 ) :

Data collected from experiments where total Mn2$and total UDP-galactose concentrations had been varied systematically could now be compared with the predictions of the rate equations in terms of A4 and A , , since the experimental conditions allowed a proportionality between M and the total concentration of Mn2+. Experiments were also carried out with the concentrations of two of the three reactants, M , A and C, varying in such a way that either the ratio or the product of the concentrations was a constant. Different values of this constant were then chosen. Kinetic runs were set up with the appropriate total concentrations ( M o and A,) so as to give the desired values of M , A and C. The selection of a constant ratio or a constant product was determined by the relationship shown by the two concentrations in Eqn (5). For example, M and A were varied so that A4 x A remained constant, while M and C were varied so that M / C remained constant. Replacement of the two concentration variables in the rate equation by a single concentration variable and a parameter representing the value of the ratio or product produced a modified rate equation which in some cases possessed characteristics enabling it to be readily distinguished from the rate equation of a closely similar mechanism. In general, five values of the concentration of one of the reactants were chosen and five values of the ratio or product. There were thus 25 values of the concentration of the second reactant. The concentration of the third reactant was determined by the value chosen for the ratio or product. The data were examined by means of double-reciprocal plots of 0 - l against the reciprocal of one of the varying reactant concentrations. Lines could be drawn on the graph joining points corresponding to a fixed concentration of the third reactant. The appropriate rate equation in terms of M , A and C was then fitted to the data, and the experimental variance noted, as described above. RESULTS Characterisation of the Enzymes

+

(3)

The purified bovine milk enzyme gave a single band on dodecylsulphate/polyacrylamide gel electrophoresis, with an apparent molecular weight of 54000. The purified bovine colostrum enzyme showed two bands corresponding to apparent molecular weights of 54000 and 42000. The lower molecular weight band

Role of Manganese Ions in Galactosyl Transferase

182

was not always present after rechromatography on cr-lactalbumin-Sepharose, and such preparations of greater homogeneity were used for the kinetic work. Storage of colostrum preparations without serum albumin produced inactive components of apparent molecular weights 106000 and 86000 which may be dimers, Occurrence of dimers and the heterogeneity of the enzyme from milk and colostrum have been discussed by Powell and Brew [5]. From the work of these authors, the true molecular weights of the active species are 50000 and 41 000. All the present experiments were carried out with the 50000-M, component. Under the conditions of the standard enzyme assay, the purified enzyme samples were found to have specific activities of 2 nkat mg-' after addition of bovine serum albumin, and approximately 5 pg were used in each 100-pl reaction mixture. When MnC1, was omitted from the standard assay, a velocity was measured in terms of production of l4C-labe1led product which corresponded to a concentration of Mn2+of about 5 pM. and was not attributable to nonenzymic breakdown of UDP-galactose. Such values were 103-fold lower than those produced in the studies on the effect of added Mn2+ in which concentrations of up to 40 m M were taken, and so any correction to either Mn2 concentration or rate was considered negligible and therefore unnecessary.

Association Constantsj i w MnZ w,ith Mops, UDP-galacto~rand U D P +

Titration of Mops in the presence of MnCl, at 22 "C gave an association constant of 3.0 M for the Mops anion interaction with Mn2+. At pH 7.4, the apparent association constant is therefore 1.9 M-', using a value for the pK, of Mops of 7.16, determined by titration without MnCl,. From the ESR studies at 18 "C, there appeared to be a 1 : l interaction between Mops and Mn2+ and between UDP-glucose and MnZ+. The respective association constants (uncorrected at pH 7.4) were 1.96 M-' and 58.3 M-', the latter value being taken to apply to UDP-galactose as well. The best fit to the UDP data corresponded to a value for n, the number of Mn2' bound per UDP, of 2.4 and a value for K (Eqn 1) of 3200 M -' At 37 C the relative decrease in the Mn2+ signal due to complex formation was the same as at 18 'C showing that there was no appreciable diff'erence in the values of the association constants at these two temperatures, The values reported above were therefore used in subsequent calculations relating to the kinetic experiments at 37 "C, the value of 1.96 M being taken for Mops, in good agreement with, but more accurate than, the value determined by potentiometric titration.

Some experiments were also carried out to estimate the effect of the galactosyl transferase protein and the bovine serum albumin on the concentration of free Mn2+. The effect was such that it could be ignored under the conditions of the kinetic experiments where the protein concentrations were considerably lower. KINETIC STUDIES

Preliminary experiments confirmed that initial velocities were being measured and that they were proportional to enzyme concentration. The velocities were unaltered by variations in Mops concentration over the range 10 - 50 mM.

Basic Mechanlvm Systematic variation of the concentrations of total UDP-galactose and GlcNAc in a manner similar to that described by Khatra et al. [4] for the human milk enzyme produced a set of apparently parallel lines when double-reciprocal plots were made with each line corresponding to a fixed concentration of the second substrate. By repeating the experiments in the presence of 0.25 mM UDP-glucose, a competitive inhibitor with respect to UDP-galactose [4], the lines were shown to intersect in the same point, for both the bovine milk and bovine colostrum enzymes (Fig. 1). This result is consistent with a mechanism wherein UDP-galactose adds to the enzyme before GlcNAc and no product is released in between [4].

-20L Fig. 1. Ef$>ct of' GlcNAc cotzcmtrution.y on thc inirial velocitj. o f ' the reaction at drferent ,fixed total concentrations of UDP-galuctose und u fixed total concentration of M n 2 + ( l o m M ) in the presence of 0.25 mh4 UDP-glucose. The enzyme was prepared from colostrum, 0.32 mM, and the total concentrations of UDP-galactose uere: (0) (A) 0.16 mM. (0)0.107 mM. (A) 0.080 mM. (H)0.064 mM. The lines were drawn from the computer fit of Eqn (6) with M representing UDP-gdlactOSe and A, GlcNAc

A . D. Tsopanakis and D. G. Herries

183

When the concentration of total UDP-galactose was varied at several fixed concentrations of total Mn2+, the data could be fitted by Eqn (6) which is derived with the assumption that the interaction of Mn2+ with the enzyme is not at equilibrium. This result is in contrast with that of Morrison and Ebner [181. VMA U= (6) Ki,Ka K,M+ K,A MA

+

40

30

+

where M and A are the concentrations of Mn2+ and UDP-galactose respectively, V is the maximum velocity reached at infinite concentrations of both substrates, K, and K, are the respective Michaelis constants, and Kimmay be the dissociation constant of the first substrate from an enzyme complex or may have no obvious physical significance [19].

10

EfSects of High Mn2+ Concentration When the range of fixed concentrations of total MnZ+ was extended upwards within the same experiment, the new data could still be fitted by Eqn (6) but the values of the parameters differed from those in the lower range. There was a decrease in the apparent Michaelis constant for UDP-galactose at high Mn2+ concentrations, exceeding the decrease expected by extrapolation from the results at low Mn2+ concentrations. An efTect of this kind represents an activation by Mn2+, and when initial velocities were measured as a function of Mn2+ concentration at a fixed level of UDP-galactose, a downward-curving double reciprocal plot was obtained and the data could be fitted by Eqn (7): VM(K, M ) U= (7) K, i K3M f M 2

Fig. 2. Eflect qf total UDP-galactose concentrations on the initial velocity of the reaction over a wide range of concentrations of free Mn2+ and a fixed concentration of GlcNAc (10 m M ) . The enzyme was prepared from milk and the lines were drawn from the computer fit of Eqn (9). The concentrations of free Mn2+ in each line were approximately constant, and the corresponding constant total concentrations of manganese were: (0) 10 mM, (A) 5 mM, (V) 3.33 mM, (0) 2.5 mM, (0)2 mM, (A) 1 mM, (V) 0.67 mM, (m) 0.5 mM, (CI) 0.4 mM. The values of the parameters in Eqn (ll), which is equivalent to Eqn (9), were: K, the MnUDP-galactose stability constant, 58.3 M-'; KL defined as zero; V , , 110 nM s - ' ; V , , 180 nM s-'; Kim, 3.6 x M ; K a , 1.02 M ; K,,,, 2.3 x x 10-4 M ; K ~ 8.0 , x 10-5 M

where M is either the concentration of free Mn2+ or the total concentration since these are proportional within experimental error (see Materials and Methods section); V is the maximum velocity reached at infinite M , and K,, K, and K3 are adjustable parameters of the equation.

where A , represents the total concentration of UDPgalactose, V is the maximum velocity and K, the Michaelis constant. The above equations which describe the dependence of rate on total UDP-galactose concentration (Eqn 8) and on the free Mn2+ concentration (Eqn 7) can be combined to give Eqn (9) :

Efect of Total UDP-galactose Concentration

v=-

"0

5

10

15

10e3/Total [UDP Gal] (M-')

+

A wide range of both [Mn"] and [UDP-Gal] was investigated to establish the rate law for the dependence of initial velocity on total [UDP-Gal]. No departure from linearity was found in any double-reciprocal plot for either the colostrum or the milk enzyme with total UDP-galactose as the variable substrate and a fixed concentration of total Mn2+.The behaviour was thus in agreement with Eqn (8):

VMA,(Ki+ M ) . K,(K3+K4M+M2)+(K5+ K,M+M2)A,

(9)

There are seven adjustable parameters, Kl , K,, K 3 , K4, K 5 , Ks and the maximum velocity, V. As will be seen later (Eqn 1I), these can be expressed in terms of an alternative set of seven parameters, to which physical meaning can be ascribed (e.g. K,, the Michaelis constant for M). When this is done, it is also seen that a special form of the equation in which one of the alternative parameters is redundant, gives the best fit to the data, so that in fact there are only six independent adjustable parameters. Fig. 2 shows

3 84

Role of Manganese Ions in Galactosyl Transferase

the data plotted with total UDP-galactose as the variable substrate and F i g 3 with free Mn2+ as the variable substrate, Elirnincition of Mechunisms not Consistent with Preceding Rerults

A large number of mechanisms was investisated. These were considered to be variants of the basic mechanisms put forward by Khatra et al. [4] and Morrison and Ebner [I 81, i.e. a sequential mechanism involving ordered addition of M (Mn"), A (UDPgalactose) and B (GlcNAc), with a central complex of enzyme, M, A and B, isomerising to enzyme, M, P and Q, mhere P represents LacNhc and Q, UDP. The

3c

_. n

'C

0

I

n

5

I

'.O

I

1.5

10~3/ [ Mn"]

I

I

I

2.0

2.5

3.0

(M-')

Fig. 3. Flffect of wick rrrnge o f f r e e MnZ+ concentrutions on the initiul celoc,rty of the reuc,rion tit difermt total concentrations of UDP-Gul und u f i w d concentration of GlcNAc (10 m M ) . The data are the same as those in Fig.' and the lines again drawn from the computer fit of Eqn (9). The total concentrations of UDP-Gal were: (A) 0.32 mM. (V) 0.16 mM, (m) 0.107 mM, (+) 0.08 mM, ( 0 )0.064 mM

Scheme I

complex, MnUDP-Gal, is represented by C. and the complexes MnUDP and Mn2UDP by R and S respectively. C is equivalent to MA, R to MQ and S to M R or M2Q. The mechanisms considered were various combinations of the pathways shown in Scheme 1. There are two routes from substrates to products, one involving a central complex with one Mn2+ (EMAB) and the other a central complex with two Mn2' (EM,AB). Possible mechanisms which included an alternative pathway not involving Mn2+ as substrate or part of a substrate were rejected on the grounds that there is an absolute requirement for M n 2 + [ 3 ] . At the other extreme, mechanisms with alternative pathways utilising C in one branch and M then C in the other were also rejected because of the improbability of there being so great a requirement for Mn2* . Since the basic mechanism requires the addition of M before A, no mechanism was considered that had A adding before M, except as an alternative pathway in a modification of the basic mechanism, The formation of dead-end inhibitor complexes was also considered, involving combination of a reactant with either the free enzyme (competitive), the enzymesubstrate complex (uncompetitive) or both (noncompetitive). In most cases, the theoretical inhibitor was the UDP-galactose species not involved as a substrate, for example, C when A was the substrate and vice-versa. In addition, the effect of the binding of some or all of the substrates at equilibrium and of the release of some or all of the products at equilibrium was investigated in all but the most complex mechanisms. The following procedure was carried out. A rate equation in terms of substrate, product and inhibitor concentrations was derived for each mechanism, simplified by setting product concentrations (except M ) to zero, and expressed in terms of M and A,. Mechanisms were eliminated when the predicted dependence of initial rate on M and A,, did not agree with the experimental findings, namely that the doublereciprocal plot in A,, should be linear. and that high concentrations of Mn2 should activate, +

185

A. D. Tsopanakis and D. G. Herries

I

0

I

I

2

4

I

I

6 8 [UDP Gal] (M-’)

I 10

I 12

Fig.4. Effect of the concentration of free UDP-Gal on the initial velocity of the reaction at severa1,fixed uulues ofthe product [UDP-Gal] x [Mn”] and a jixed concentration of GlcNAc (10 m M ) . This is an A x M graph, with each line at a different fixed value of A x M , or of C. The enzyme was prepared from colostrum. The lines were drawn according to the computer fit of Eqn (12). Values of C were: (A) 0.15 mM, (v) 0.075 mM, (W) 0.05 mM, (+) 0.0375 mM, (0) 0.03 mM. The values of the parameters in Eqn (10) were: Vl , 160 nM s-’; V 3 , 370 nM s - ’ ; Kim,6.0 x M ; K,, 3.5 x M ; Kk defined as zero; K a , 1.07 x M; K c , 2.8 x M

A group of mechanisms that involved addition of M followed by either A or C (pathways 1 and 2 of Scheme 1) could not be eliminated. The most general rate equation for this group is Eqn (10) (see Appendix):

(10) Vl is the maximum velocity and Km the Michaelis constant for M in the pathway using A as substrate, V, and KA are the corresponding parameters in the pathway using C, and K, and K, are the Michaelis constants for A and C respectively with Kimthe dissociation constant of M from its complex with the free enzyme. This equation applies to the mechanism with the pathways labelled 1, 2, 3 and 6 in Scheme 1; in mechanisms with pathways 1 , 2 , 4 and 5 or 1,2,4 and 6, K,=O, and in mechanisms with pathways 1 , 2 , 4 and 5 or 1, 2, 3 and 5, KA = 0. Transformation by applying Eqns (3) and (4),where K is defined by Eqn (9, produced Eqn (1l),which has the same form as Eqn (9) and is thus consistent with the results described in the previous section :

Experiments with M and A at constant values of the product, and M and C, or A and C, at constant values of the ratio, were carried out. Six such experiments were considered, denoted as C / M , M / C , CIA, AIC, A x M and M x A , where the first letter of each pair signifies the substrate whose concentration was given five values and the second, 25 values. The concentration of the third substrate is automatically fixed by Eqn ( 5 ) and is constant for each line of the doublereciprocal plot. Three rather than six transformations of the rate equation in terms of M , A and C were required. Thus the transformed equation in terms of M and A for a C / M experiment was the same as that in terms of A and M for a CIA experiment, and was plotted as l / u against 1/A with each line at constant M . Similarly three rather than six types of experiment were carried out, as data in a C / M experiment could also illustrate an A x M experiment, because in both kinds, the concentrations A and C each had five values, and the concentration M , 25. The results of an A x M experiment are shown in Fig.4. The lines are described by an equation that results from the transformation of the rate equation in terms of M , A and C to one in terms of C and A only, so that I/v is plotted against 1/A with each line at constant C. Data for an AjC experiment produced a family of straight lines similar to those shown in Fig.2, when l / v was plotted against 1 / A with each line at constant M . The mathematical form of this equation in terms of A is always the same as that of the M , A , equation in terms of A , , because both C and A are proportional to A , when A4 is constant (from Eqns 3 and 4). The group of mechanisms identified above as being consistent with the experimental results can be narrowed by considering Fig. 4. The appropriate transformation of Eqn (10) in terms of A at constant C that applies to Fig. 4 is Eqn (12): v=-

C(V,Ka/Kc+ VIA I

~-

Ka(1+C/Kc)C+(KKimKa+[1+KK~]C)A+KK,A2’ (12)

If K, = 0, the denominator A’ term is absent, and this equation predicts that the graph of l j v against 1 / A will be a rectangular hyperbola with asymptotes parallel to the axes of the graph. Since the lines in Fig.4 do not show this property, mechanisms with Km=O must be eliminated. A consideration of the corresponding plot of the data against 1/M, and of other data for an M x A experiment, supported this conclusion.

186

Role of Manganese Ions in Galctosyl Transferase UDP-Gal

I

MnUDP-Gal

GlcNAc

1

GlcNAc

LacNAc

LacNAc

MnUDP

C Mn2' t

MnUDP

Scheme 2

Final Clzoice of Mechanism by Statistical Analysis

To distinguish between the remaining two mechanisms, the rate equation for each was fitted to the data, and the statistical variance examined. Eqn (10) is the rate equation for the mechanism with pathways 1, 2, 3 and 6 of Scheme 1, and the similar equation for the mechanism with pathways 1, 2, 3 and S has the denominator KA term absent. In every set of experimental results, the variance in fitting this second equation with six adjustable parameters was lower than in fitting Eqn (10) with seven adjustable parameters, notwithstanding the expectation that the more parameters available for adjustment, the better the fit should be, The mechanism comprising pathways 1, 2, 3 and 5 of Scheme 1 was thus found to be the mechanism most consistent with all the experimental results, and is shown in Scheme 2. It should be noted that the kinetic data were analysed assuming equal weights for the velocity measurements. Plots of the residuals (calculated velocity minus experimental velocity) against experimental velocity [20] showed bands of points contained within the values +20 where CT is the square root of the experimental variance, defined as the sum of squares of the residuals divided by the difference between the number of experimental points and the number of parameters being estimated. The appearance of the points in a parallel band about the 2: axis suggests that the weighting assumption was correct, and the random distribution of positive and negative residuals supports the correctness of the equation used in fitting the data. The values of the parameters are reported in the figure legends but it may be noted that only the value for the dissociation constant of Mn2'from the E-Mn" complex, Kim.is independent of the concentration of GlcNAc (see Appendix). Values of Kim ranged between 0.4 and 7.5 mM in different experiments, to be compared with literature values of 1.4 mM [4] for the human milk enzyme, about 3 mM for the bovine colostrum enzyme [5], 1.6 mM for the bovine milk enzyme cross-linked covalently to a-lactalbumin [21], all under the same conditions as the present work, and 1.4 mM for the bovine milk enzyme [18] under different experimental conditions.

DISCUSSION The enzyme species from bovine colostrum and bovine milk used in the present work both had molecular weights of 50000 and may therefore be presumed to represent the undegraded form of galactosyl transferase originally present in the Golgi membranes [5]. Unlike Powell and Brew [S], we found evidence of a species with molecular weight 41 000 in colostrum, but all the experiments reported here were carried out with the larger component. We have assumed that the same molecular weight species from colostrum and milk will have the same kinetic properties. The results may be compared with those obtained by Khatra et al. [4] using the human milk enzyme, also of molecular weight 50000. The results described in this paper are in agreement with the basic mechanism of Khatra et rrl. [4] and Powell and Brew [5]. The presence of a fixed concentration of a competitive inhibitor (UDP-glucose) enabled data to be obtained (Fig. 1) that confirmed the sequential nature of the mechanism involving the addition of UDP-galactose followed by GlcNAc. In a ping-pong mechanism, the graph of the results in the presence of UDP-glucose would have remained as a set of parallel lines. Confirmation of this aspect of the mechanism by other kinetic means has already been reported [14]. The effect of the pair of substrates, UDP-galactose and Mn2+, on the velocity was in agreement with the previous reports [4,5] but at variance with the result of Morrison and Ebner [18] for the bovine milk enzyme, under difl'erent conditions of pH, buffer and temperature, and later confirmed by Geren et al. [22]. It has been suggested [ S ] that the enzyme used in the earlier studies [IS] may have contained a significant proportion of the lower molecular weight species which may possess different kinetic properties, but this would appear to be refuted by the later results [22]. The basic mechanism is therefore an ordered addition of Mn 2 +, UDP-galactose and GlcNAc to the enzyme, without release of Mn2' solely as the free ion. The activating effects of higher concentrations of Mn2+ were investigated, and the elimination of a large number of mechanisms that were modifications of the basic mechanism was made possible by the fortuitous circumstance in which the concentration of free Mn2+ in the reaction was proportional to total manganese concentration. The remaining few mechanisms were examined with substrate concentrations taken at a constant ratio or constant product. It was necessary to proceed in this way because the independent variation of the concentrations of two substrates with all other concentrations being held constant was impossible, owing to the relationship between M , A and C expressed by Eqn ( 5 ) .

A. D. Tsopanakis and D. G. Herries

187

It is interesting that the final mechanism (Scheme 2) was found to be a modification of the basic mechanism of Khatra et al. [ 4 ] ,with release of Mn2+ as MnUDP in the branch in which UDP-galactose is substrate. The corresponding mechanism of Morrison and Ebner [ 1 8 ] , in which Mn2' is released as the free ion, is part of the mechanisms that have Km=O, both of which were unequivocally eliminated. Our suggested explanation [23] of the apparent equilibrium binding of MnZ+ found by Morrison and Ebner [ 1 8 ] would therefore seem to be untenable. The values of the kinetic constants support the view that at high Mn2 concentrations, the formation of MnUDP-galactose provides an alternative substrate to act as galactosyl donor with an increased maximum velocity ( V , in Eqn 10). In a number of experiments not reported here, and in the results in Fig.2 and 4, the maximum velocity in this branch of the mechanism is about twice that in the branch using UDP-galactose as substrate. It should be remembered that this ratio and the values of the Michaelis constants are dependent on the GlcNAc concentration (see Appendix). Comparison of the Michaelis constants does not lead to a conclusion, as their ratio in most experiments was about 1. It would therefore seem that the activation by Mn2+ at 10 mM GlcNAc is through a faster transglycosylation step in the mechanism than through preferential binding of MnUDP-galactose to the E-Mn2+ complex. The enzyme species involved in these experiments must clearly be the Mn:+/Mn;,+ enzyme described by Powell and Brew [ 3 ] , and none of the kinetic phenomena they observed at limiting

Mn2+ concentrations would be expected in the present study where the concentrations were in a much higher range.

APPENDIX Rate Equation f o r the Mechanism of Scheme 2

The rate constants are defined as shown in the scheme below: A

P

R

+

\

EMC

(EMBC,EMPR)

EMR

EM

b

I

I

I

klllk12

k13 1*14

*15Ik16

k171k18

kllk2

C

B

P

R

M

Scheme 3

The following combinations of rate constants are defined :

u = k, + k7 b = k14 k , , c = k4k,,k,,a d= k,k,k,

+

+ k,k,k,zb

=k13k15k17.

The initial velocity equation may be derived from the complete equation by setting P and R to zero :

k,k,d(k,,k,,b+eB)BMA +k,k,,e(k4k,a+dB)BMC k2(k4k,k,2k,7ab+k,k, ,cB deB2) kI(k4k,k,,kl7ab k,k,,cB deB2)M k3d(k,,k,,bB eB2)A k , k3(k,k,,k,,ub k17[k9k13k1 k,k12b{ k , + k,} IB k,e [k, k,]B2)MA + k l k~l(k4k9ki7ab + k9[k4k13a { k 1 5 + k17) + k5k7ki 7b 1B + kl3d[ki 5 + k17 IB2)MC

L'=

+

+

+

+

+

+

+

+

+ +

+

By defining the following kinetic parameters, Eqn (10) with the denominator C term absent may be derived:

v,

+

d(k,,k,,b eB)B k9k,2k17ab+Ic,7(kyk,3kl~a+k5k,2b [k7+k,l)B+k5e(k7 +k9)B2

=-

.____ ._

188

A. D. Tsopanakis and D. G, Herries: Role of Manganese Ions in Galactosyl Transferase

We are grateful to Dr P F Knowles for allowing us to use hls electron Ypin resonance spectrometer

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1432-1440. 2. Brodbeck. li, Dcnton, W. L,,Tanahashi. N. & Ebner, K. E. (1967) .I Bird Chern. 242. 1391-1397. 3. Pornell, J . T. & Brew. K, (1976) J . Biol. Chem. 251, 3645-3652. 4. Khatra, B. S . Herries, D. G. & Brew, K. (1974) Eur. J . Biochem. 44, 537- 560 5. Powell. J. T. & Brew. K. (1974) Eur. J . Biochem. 48, 217-228. 6. Aschail'enburg. R. & Drewry. J . (1957) Biochern. J . 65, 273-

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A. D. Tsopanakis, School of Molecular Sciences, University of Sussex. Falmer, Brighton. Great Britain, BNI 9QJ D. G . Herries*. Department of Biochemistry, University of Leeds, 9 Hyde Terrace, Leeds. Cireat Britain. LS2 9LS

*

To whom correspondence should be addressed

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