THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 235, No. 5, May 1960

Printed in U.S.A.

Mutarotase from Penicillium notatum

II. THE MECHANISM OF THE MUTAROTATION REACTION*

RONALD BENTLEY AND D. S. BHATE~

From the Department of Biochemistry and Nutrition, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania

(Received for publication, June 10, 1959)

There are relatively few enzyme-catalyzed reactions directly involving an asymmetric carbon atom, which result in inversion of configuration. Three such enzymes are maltose phospho- rylase, cellobiose phosphorylase, and ,&amylase, which catalyze reactions of the following type with replacement of a group X by a different group Y.

“i a

b-C-X --f b Y- -b I c c

Such reactions have been considered to proceed by a single displacement mechanism (2). Other enzymes catalyze race- mization reactions of the type:

i i b-C-X + X-C-b

I I c c

References to such stereoisomerases have recently been summa- rized (3). The mechanism of these reactions is not well under- stood. Some of the pyridoxal phosphate-requiring amino acid racemases probably proceed through a Schiff base intermediate so that a single displacement mechanism is unlikely. Single displacement mechanisms are probably not involved in the racemization of hydroxy-acids for which a dehydrogenation step is necessary, and are ruled out for the diphosphopyridine nucleotide-requiring uridine diphosphogalactose-4-epimerase. In some other cases, there is insufficient evidence to determine reaction mechanisms with precision.

One such racemization reaction which has been extensively studied by chemical methods is the mutarotation of glucose; the spontaneous, acid- and base-catalyzed mutarotations are believed to involve the intermediary formation of the open chain aldehyde. With purified preparations of mutarotase avail- able (4), it appeared possible to decide whether the enzymatic reaction represented a single displacement mechanism or was analogous to the chemically catalyzed reactions.

EXPERIMENTAL

Materials and Methods

General-cr-n-Glucose was the standard dextrose sample ob- tained from the National Bureau of Standards. a-n-Galactose

* A grant from the United States Public Health Service (A-725)

was recrystallized as described by Bates et al. (5). 2-Hydroxy- pyridine (Sapon Laboratories, Valley Stream, New York) was recrystallized twice from benzene, m.p. 108’. Histidylhistidine was obtained from Mann Research Laboratories. Mutarotase from Penicillium notatum was prepared as described by Bentley and Bhate (4).

Polarimetric 1Methods--Optical rotation measurements were made in a jacketed 2-dm tube of 3 mm diameter, usually at 24”; the tube, solvents, and all glassware were brought to tem- perature equilibrium for at least 15 minutes before solution of the sugar. After thorough mixing the sugar solution was drawn into a 5-ml hypodermic syringe attached to a 24 cm length of polyethylene tubing of internal diameter, 0.062 inches.’ The plastic tubing was inserted through an open end of the polarim- eter tube, pushed all the way down to the closed end, and the sugar solution was then “injected” into the polarimeter tube with a simultaneous withdrawal of the plastic tube. The micro- polarimeter tube could thus be filled without shaking to remove bubbles, and readings were routinely obtained within 2 min- utes from the time of solution of the sugar. Equilibrium ro- tations were determined after allowing the solutions to stand overnight.

For the reversible mutarotation reaction,

kl c? ;===? P, k2

the mutarotation coefficient

k = k~ + kz = l/t log s ; m

ro is the optical rotation at zero time, rt the rotation at time t, and r, is the final equilibrium rotation. k was determined from the plot of log10 rt - r, against time and unless other- wise stated, all coefficients are expressed in logarithms to base 10 and reciprocal minutes. The following symbols have been used:

k apont = measured mutarotation coefficient in water alone. If necessary to distinguish between Hz0 and DzO, the sub- script notation will read, “spont in HzO,” or “spent in D,O.”

k obs = measured mutarotation coefficient for a given cata-

made this study possible, and is gratefully acknowledged. A preliminary report of some of this work has already been given (1).

t Present address, Antibiotic Research Centre, Hindustan Anti- biotics (Private), Ltd., Pimpri, India.

1 Intramedic polyethylene tubing, PE 205, Clay-Adams, Inc., New York.

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1226 Mutarotase from P. notatum. II Vol. 235, No. 5

lyst in water (HgO) unless further modified to indicate the use of DZO.

k, = catalytic coefficient (mutarotation coefficient for molar solution) for a given catalyst z.

ka, kn = catalytic coefficient for a given catalyst, the nature of which is known from the context, in Hz0 and DzO, respec- tively.

The ratio i&/i& has been determined in some cases from the equation

ka/kD = (kobs inq~ - kspont in H,O)/(kobs in D,O - kspont in D,O )

where the two values of k&s in Hz0 and DzO were determined at the same catalyst concentration. In other cases a graphi- cal evaluation of kH and lcn was made by determining the slope of the plot of values for k&s against catalyst concentration in both solvents.

Isotopic Methods-DzO was obtained from Stuart Oxygen Company, San Francisco and the Liquid Carbonic Division of General Dynamics Corporation, San Carlos, California, on al- location of the United States Atomic Energy Commission. The material contained greater than 99.5yo deuterium and will be referred to as 100% DzO. n-Glucose-l-018, prepared by heat- ing ordinary glucose in HzO1s solution for 18 hours at 100’ (6), was kindly supplied by Dr. D. Rittenberg. We are also very much indebted to Dr. Rittenberg and Miss L. Ponticorvo for the mass spectrometric analyses for D and O18.

Previous preparations of glucose-l-D had used carrier glu- cose-1-H (7) or yielded a material with a wide melting range (m.p. 141-149”) (8). An improved preparation of this mate- rial was devised. The method was based on the reduction of d-gluconolactone used in the preparation of glucose-l-Cl4 (9) and required only limited amounts of DzO. All reagents used were anhydrous and were carefully dried before use to mini- mize the dilution of DzO as far as possible. n-Glucono-d-lac- tone (5 g), sodium acid oxalate (16.5 g), and oxalic acid (11 g) were put in a Waring Blendor in the cold room. Ice-cold DzO (20 ml) was added and the Blendor was run briefly to mix these reactants. Then 200 ml of anhydrous ether, cooled to -5”, and 65 g of ice-cold, 5% sodium amalgam in the form of “shot” (10) were added. The mixture was homogenized for 7 minutes, when a further 5 ml of ice-cold DzO were added, and the agitation continued for a further 8 minutes. After about 5 minutes, the temperature had risen to the boiling point of the ether. After cooling, the ether was decanted off, and the residue was centrifuged. The clear aqueous supernatant was distilled under reduced pressure in a closed system, about 6 ml of DzO being recovered. The residual salts were washed three times with methanol, mixing each time in the Waring Blendor. The combined methanol washings were evaporated to a thin syrup; this syrup and that obtained after removal of the DzO from the original supernatant were dissolved in 50 ml of water and treated with N NaOH until a permanent pink color to phenolphthalein was obtained. The solution was evaporated to a very thin syrup and 45 ml of methanol added slowly so that a solid precipitate formed. This precipitate was removed by centrifugation, and was washed with methanol (10 ml). The combined supernatant and wash fluid were evapo- rated to a volume of 10 ml, and water, 5 ml, and methanol, 75 ml, were added. A partly crystalline solid separated on stand- ing and the opalescent mother liquor was decanted and evapo- rated to a syrup. The syrup was treated three times with water (20 ml) re-evaporating to a syrup each time to complete the

exchange of labile deuterium in O-D linkages. The syrup, in water, 20 ml, was passed through a column (18 mm in di- ameter) containing at the lower end Amberlite IR 120 (H) (1.5 cm) and above this, Amberlite MB 2 (22 cm). Elution with water gave a still acid solution (400 ml) which was evapo- rated to a volume of 20 ml, before repetition of the resin treat- ment. The final eluate, evaporated under reduced pressure to about 4 ml, was treated with methanol (6 ml) and then drop- wise with isopropanol (5 ml). Crystallization took place quickly on seeding with a very little anhydrous ac-D-glucose-l-H. After standing overnight, the crystalline mass was filtered and washed with ethanol; yield, 2.73 g. On standing of the washings, a second crop slowly separated (0.24 g). Measurement of optical rotation indicated that the first crop material was a mixture of o( and p anomers, in almost the proportions of the usual equilibrium mixture. The second crop material, however, was almost pure o( anomer, m.p. 14G-147”. The first crop material was obtained as the o( anomer, m.p. 147”, by a slow recrystal- lization from methanol-isopropanol. The equilibrium rotation of recrystallized glucose-l-D was determined accurately from the average of nine determinations; [ali = +52.65 f 0.19’. The substitution of H by D in this case apparently has no detectable effect on the optical rotation. The material was found on analysis to contain 1.1 atoms of deuterium per mole- cule.

RESULTS

Reaction Mechanism-Although it is probable that the spon- taneous and acid-base catalyzed mutarotation of glucose pro- ceeds through the intermediate aldehyde structure, the possi- bility of dehydration or dehydrogenation mechanisms with mutarotase could not be excluded a priori. Experiments were therefore carried out to determine whether 01* was lost from glucose-l-O’* during mutarotation in HxO*@ or whether H was lost from glucose-l-H during mutarotation in DzO.

1. Mutarotation of Glucose-l-018 in HzO-Thirty-five milli- grams of n-glucose-l-018 (4.45 atom y. excess 018) were dis- solved in 1 ml of deionized water, and left, for 3 hours at 21”; in a second tube, 125 pg of mutarotase in 0.05 ml of Hz0 were also added. The solutions were lyophilized, the residues dis- solved in 1 ml of water, and again lyophilized. The dry resi- dues were taken up in 1 ml of hot ethanol and filtered; a further 1 ml of ethanol was used to wash the filter. The filtrate was evaporated to about 0.5 ml, seeded with a very little glucose, and allowed to crystallize, first at room temperature for 2 days, and then in the cold room overnight. The crystallized glu- cose was filtered, washed with 3 to 4 ml of ice-cold ethanol, and dried over phosphorous pentoxide in a vacuum. The re- covered glucose was 25.7 mg in the control experiment, and 28.1 mg in the experiment with mutarotase. Both samples had m.p. 147”. 01* analyses of the recovered samples gave the fol- lowing values in duplicate determinations. Spontaneous muta- rotation, 4.10, 4.03 atom ‘% excess O1*; enzyme-catalyzed muta- rotation, 4.05, 4.08 atom y0 excess O1*.

2. Mutarotation of Glucose-l-H in DzO-In these experiments, 100 mg of oc-D-glucose-l-H were dissolved in 5 ml of DzO or in the same volume of DgO containing 1 mg of lyophilized mutarotase. Mutarotase was known to be stable to lyophili- zation. After standing at 21” for 3 hours the solutions were lyophilized; the residues were dissolved in Hz0 (2 ml) and again lyophilized. This operation was repeated four times, be- fore crystallization of the glucose, to ensure removal of any

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1 I I I I I 1 I 20 40 60 80 100 120 140

Time (minutes) FIG. 1. Mutarotation of cu-n-galactose in Hz0 and D20. Curve

A, DsO alone; Curve B, DzO plus mutarotase, 19.2 pg per ml; Curve C, Hz0 alone; Curve D, Hz0 plus mutarotase, 19.2 pg per ml. rL is the observed optical rotation at time t, r, the final equilib- rium rotation. In each case the galactose concentration was 2%. The experiment with mutarotase in D,O was carried out with a previously lyophilized preparation as described in the text. The initial deviations from linearity due to the rapid phase of the galactose mutarotation do not show well on these condensed plots. They are most obvious in the two D20 experiments shown in Curves A and B. Many more points than can be conveniently shown were obtained for the early, rapid phase of the mutarota- tion.

labile D. The duplicate deuterium analyses were as follows; spontaneous mutarotation, 0.052, 0.038 atom ‘% excess D; en- zyme-catalyzed mutarotation, 0.047, 0.037 atom ye excess D.

Another possible enzymatic mechanism was suggested by the observation of Swain and Brown (11) that 2-hydroxypyridine was a bifunctional catalyst for the mut.arotation of tetramethyl- glucose in benzene solution. If mutarotase were a bifunctional mutarot,ating catalyst, 2-hydroxypyridine might be an enzyme inhibitor. Mutarotation coefficients were therefore deter- mined for 2 y0 glucose solutions, both with and without 0.01 ye 2-hydroxypyridine, and with and without mutarotase. The hy- droxypyridine had no effect, either on the spontaneous or on the enzyme-catalyzed reaction.

Kinetic Experiments-In view of the known effect of solvent DzO in decreasing mutarotation rates (12), and of the variation of the k&n ratio with the nature of the catalyst, the spon- taneous and enzyme-catalyzed mutarotations of cr-n-glucose-l- H, a-n-glucose-l-D, and a-n-galactose-1-H were compared in Hz0 and DzO. For experiments in 100% DzO, an appropriate volume of enzyme solution in HaO, usually 0.3 ml, was lyophil- ized; great care was taken that none of the flaky, dried prep- aration was lost. After further drying overnight with calcium chloride in a vacuum desiccator at 0’ the residue was redis- solved in a given volume of 100% DzO. Typical kinetic plots

are indicated in Fig. 1 for the spontaneous and enzyme-cata- lyzed mutarotation of cr-n-galactose in both Hz0 and DzO; with this sugar mutarotation coefficients were determined for both the fast and slow mutarotations as described by Isbell and Pigman (13). The results of these experiments are given in Tables I (glucose-l-H and glucose-l-D) and II (galactose-1-H).

In the experiments just described, the mutarotase was usually

TABLE I

Mutarotation coeficients for a-o-glucose-l-H and or-D-glucose-l-D in Hz0 and 100% DzO

The measurements with glucose-l-H were made at 23” and with glucose-l-D at 24”, with 2% substrate concentrations. In the latter case, the values given are averages from two determina- tions. The coefficients in HzO, corrected to 20” with the same value for the activation energy in each case are 62.4 X 1OP (glu- cose-1-H) and 63.2 X lO+ (glucose-l-D); the accepted value for glucose-l-H is 63.2 X 1P (5). The enzyme used was a prepara- tion of Fraction F which in the bromine oxidation assay (4) had, originally, an activity of 250 pmoles net P-glucose per mg protein per 20 minutes. A mutarotation coefficient determined at that time gave a value of 213.2 X lo-* (25 pg protein per ml). The en- zyme had been stored for several months before the glucose-l-H experiments and again before the glucose-l-D experiments. In each case there had been some loss of activity, and consequently, it appears that there is a large difference in the coefficients of glucose-l-H and glucose-l-D. Subsequently, kobs was measured for the two substrates under identical conditions with a different mutarotase preparation (54.0 rg per ml); the coefficients were respectively 219.3 X 1OP and 211.3 X 1OP for glucose-l-H and glucose-l-D and it is apparent that there is little or no difference between the two compounds.

Substrate

Glucose-l-H Glucose-l-H Glucose-l-H Glucose-l-H Glucose-l-D Glucose-l-D Glucose-l-D Glucose-l-D

Mutarotation coefficient

!&ml Hz0 0 DzO 0 Hz0 25 DzO 25 Hz0 0 DzO 0 Hz0 24 DzO 24

lo4 k&s, min-’

knh

3.56 (Water)

1.86 (Mutarotase)

3.78 (Water)

1.80 (Mutarotase)

TABLE II

Mutarotation coejicients for a-D-galactose in Hz0 and 100% DzO All measurements were made at 24” and galactose concentra-

tions of 2%. For the fast mutarotation reactions, the coefficients are expressed as lo3 kobs, min-I, and for the slow reactions as lo4 kobs, min-I. The coefficients for galactose in water alone, corrected to 20”, are 77.3 X lC@ and 79.6 X 1P min-I, respec- tively, for the fast and slow reactions. The literature values are 79.0 X 1O-3 and 80.3 X lO+, respectively (5).

P Fast r ! mutaro-

Solvent e tation 22 coeffi-

s cient

-~__

i Slow mutaro-

kEjkn for fast reaction tation coeffi-

kE/kD for slow reaction

cient

3.76 (Water)

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1228 Mutarotase from P. notatum. II Vol. 235, No. 5

TABLE III Mutarotation coegicients for glucose-l-H and

galactose in Hz0 and O$.S% D20

In these experiments, 100 mg of substrate were weighed into a 5-ml volumetric flask. Four milliliters of 100% DzO were added, followed by 0.37 ml of mutarotase solution in H20. The volume was then made to 5 ml with more 100% DzO and after thorough mixing, optical rotations were determined in the usual way. The final mutarotase concentration was 23.7 pg per ml. All determi- nations were made at 24”. The coefficients for galactose are those for the slow mutarotation only. The kn/kn ratios have been cal- culated with the values for spontaneous mutarotation in Hz0 and 100% D20 given in Tables I and II.

similar determination of the k&n ratio was carried out in 92.6% DzO (volume for volume) with a-n-glucose-I-H and a-n-galac- tose and accurately measured volumes of the same mutarotase preparation in Hz0 used in the earlier work. The results of these experiments, shown in Table III, gave a value of 1.73 for glucose and 1.47 for galactose. In a further experiment, a graphical determination of the k&n ratio with glucose was carried out with the use of three different mutarotase concen- trations in Hz0 or 94% DzO (volume for volume). This graphi- cal determination (see Fig. 2) was probably the most accurate and gave a value of 1.85 for the ratio in 94% D20; this agreed well with the value of 1.86 obtained in 100% DzO.

Seltzer et al. (14) have discussed the difficulties of assessing Substrate Solvent Mutarotation

coefficient ktxh DzO effects in enzymatic reactions and have raised the ques- I , I

I 1 10’ kobs, ntid 1 tion whether a comparison in Hz0 and DzO is properly made under identical buffer conditions or under conditions where pH = pD. They have suggested that until a mechanism is thor- oughly understood, the safest comparison is probably that be- tween the rates on the two plateaus, at the maximum rates. A further consideration in a reaction with a high spontaneous rate, as is the case in mutarotation, is the need to work under conditions which minimize the spontaneous rate. In the pre- ceding experiments it had been considered desirable to work in unbuffered systems to avoid the further complication of a significant catalytic contribution of the buffer itself.

For the spontaneous mutarotation in dilute Hz0 buffer solu- tions, an almost constant minimum mutarotation rate was ob- served from pH 2.37 to 6.91 by Isbell and Pigman (15). In this work, the range from pH 4.6 to 6.45 was covered by potassium hydrogen phthalate buffers and from 6.43 to 6.91

Glucose Glucose Galactose Galactose

Hz0 92.6% DzO

Hz0 92.6’3& DzO

1.73 (Mutarotase)

1.47 (Mutarotase)

“0 0

- t /

by o-nitrophenol buffers. With the use of 0.005 M phosphate buffers we have observed a more pronounced rise in the muta- rotation rate at pH > 6.1. Solutions of 2ye glucose or galac-

x ui

tose in Hz0 have pH = 5.7, so it is clear that an unbuffered

i//l//i’ spontaneous determination at pH 5.7 is well within the mini- mum plateau.

For the enzymatic reaction, a pH optimum of about 5.8 was indicated by Levy and Cook (16) although measurements at higher pH were not made. We have observed with our prep- arations that there is a somewhat decreased activity beyond pH 6.0, amounting to a 13% reduction at pH 6.7. Our mu- tarotase measurements in Hz0 alone at pH 5.7 were there- fore made within the pH maximum range of the enzyme.

I I I In 2% DzO solutions, glucose and galactose showed a pH 20 40 6C value measured with the glass electrode of 6.5. With the cor-

Protein, pg. /ml. rection of +0.4 to convert to pD (17), the sugars in unbuffered D,O solutions were therefore at pD 6.9. With 0.005 M phos- phate buffers, we have observed that the pD minimum plateau for spontaneous mutarotation extends to at least pD 6.8 or 6.9. With mutarotase, the same mutarotation coefficient was observed at pD 6.3 and 6.8. The experiments in unbuffered DzO solutions at pD 6.9 were therefore within the minimum plateau for spontaneous mutarotation; for the enzymatic reac- tion, there may at most have been a small error if the pD op- timum shows a slight fall at pD 6.9.

FIG. 2. Determination of k&n ratio in 94% DtO (volume for volume) for the mutarotase reaction with a-n-glucose-l-H. l - -0, experiments in HzO; A-A, experiments in 94yo DzO. The conditions are those described in the text. The enzyme used was a later sample of Fraction F than that described in Table I.

used within 30 minutes after solution in DzO. To investigate the possibility that a different result might be obtained after a longer period for equilibration with DzO, a comparison of the rates was made with enzyme stored overnight at 0” in Hz0 and DzO solutions, or as the dry solid. The results of this experiment gave lcn/kn values of 1.97 (freshly dissolved enzyme) and 2.12 (solution stored overnight) indicating that such equi- libration had no significant effect.

A further determination of the JCn/kn ratio was made at pH 6.1 and pD 6.3, with 0.005 M phosphate buffer solutions. Both of these values are within the pH or pD optimum range for the enzyme and within the pH or pD minimum for the spon- taneous reaction; in addition they are at conditions where pH

In view of possible errors in the lyophilization process, a = pD. With two levels of enzyme concentration, the ratio

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160-

. ioo- :

2 80-

60-

I I I I _ 40 80

Protein, pg./ml.

FIG. 3. Determination of k&n ratio for mutarotase catalysis in buffered solutions. In these experiments, 0.005 M phosphate buffer solutions were used; buffers in DZO solution were prepared by evaporation of a given volume of Hz0 buffer, followed by dry- ing and re-solution in the same volume of DzO. The enzyme preparation was a sample of Fraction F which had been kept for a prolonged period of time in a deep freeze, and was the least ac- tive of the preparations used in the present work. The pH of the experiments in Hz0 buffer (0-O) was initially 6.2 falling to 6.0 over 45 minutes; in the DzO buffer (A-A) the initial pH was 5.9 (pD = 6.3) falling to 5.6 (pD = 6.0) over the same period. The reason for the decline in pH with this mutarotase sample is not known; however, the plot of log10 rt - rW against time was rigidly linear in each case for at least 45 minutes. A possible reason for the observed acid production would have been the presence of some glucose oxidase; however, using the same amounts of en- zyme, this enzyme could not be determined in manometric experi- ments. In the DzO experiments, 4 ml of 100% DzO was added to 100 mg of a-glucose in a 5-ml flask. Either 0.2 or 0.4 ml of en- zyme in Hz0 was added and the volume was finally made to 5 ml with 100% DzO. The experiment with 0.2 ml enzyme (= 40 pg protein) was therefore in 96% DzO and with 0.4 ml enzyme (= 80 rg protein) in 9270 DzO.

was determined graphically from the results shown in Fig. 3. The kn/kn ratio was 1.78 and thus agreed closely with the previous values in unbuffered solution of 1.73 (92 % DzO), 1.85

(94% D,O) and 1.86 (100 ye DrO). The values obtained for the k&n ratio with mutarotase did

not agree with those for any of the catalysts previously reported in the literature (see “Discussion”). It was, however, known from the work of Westheimer (18) that amino acids catalyze mutarotation, and that histidine at about pH 6.0 to 6.5 is ac- tually a better catalyst than the same amount of strong acid. It, therefore, seemed possible that histidine might have been involved in the active site of mutarotase, and that information on this point might be obtained from a determination of the kn/kn ratio for histidine and histidine containing peptides. These ratios were determined for n-histidine (see Fig. 4) and a preparation of histidylhistidine (see Fig. 5). The values were 3.68 and 3.85, respectively. In these experiments the Hz0 solutions were at pH 6.5 and the DrO solutions at the same pH measured with the glass electrode; the pD was, therefore, 6.9.

Molarity of catalyst

FIG. 4. Determination of kn/kn ratio for histidine catalysis of glucose mutarotation. a-0, experiments in H20; A-----& experiments in 100% DzO. To prepare the histidine solutions, equal volumes of 0.02 M L-histidine and 0.003 M L-histidine dihydro- chloride were mixed and evaporated to dryness under reduced pressure. The dried residues were redissolved in appropriate volumes of either Hz0 or DQO as required to obtain the desired, final concentrations. Such solutions all had pH values very close to 6.5.

I I I I I 0.01 0.02 0.03

Molarity of catalyst

FIG. 5. Determination of kn/kn ratio for histidylhistidine catal- ysis of glucose mutarotation. O--O, experiments in HzO; i---A,- experiments in 100% D20. 0:02 & histidylhistidine, pH 6.55, was prepared by dissolving 0.292 g of histidylhistidine in 20 ml of water, adding 0.1 N HCl (about 4.5 ml) to pH of 6.5, and making the solution to 50 ml. Aliquots of this solution were evanorated to drvness and then redissolved in either Hz0 or DzO with appropriate”volumes to obtain the desired, final Eoncentra- tions .

DISCUSSION

Since there is no significant exchange of carbon-bound hy- drogen or Cr oxygen in the mutarotase reaction, reaction mechanisms for this enzyme based on dehydration or dehydro- genation involving carbon bound hydrogen are clearly ruled out. Similar conclusions have been reached from tracer studies of the spontaneous and base catalyzed reactions (19). The 01* experiments also rule out single displacement mechanisms of the type which were once considered for the enzymatic glucose- galactose interconversion (2). Since the substitution of D for H at CL in glucose has a negligible effect on the rate of the

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1230 Mutarotase from P. notatum. II Vol. 235, No. 5

spontaneous and mutarotase catalyzed process, it is again clear derivatives. Within individual groups, there is a relationship that the C&H bond is not broken in either process and that between mutarotation coefficient, amount of reducible form at hydrogen at this position exerts no influence. The open chain equilibrium and the number of ring instability factors. For aldehyde is therefore the most likely intermediate during the mutarotase, the substrate types show the following order of interconversion of the Ci chair forms of the glucose anomers effectiveness; heptose < disaccharides < hexoses > pentoses by mutarotase, as it is in spontaneous and general acid-base (4). Mutarotase action is apparently restricted to those sugars catalysis. which normally contain only small amounts of reducible form

The free aldehyde concentration in 0.655 M glucose solution in the equilibrium solution, which have low mutarotation coeffi- is, however, only 0.0026’% (ZO), but for a series of sugars (21) cients and which have the steric requirements outlined in the the amount of reducible form in soluton (i.e. aldehyde) increases preceeding paper (4). with the number of “instability factors” (22) associated with Reactions of the following type, the individual molecular conformation. As shown in Table IV, mutarotation coefficients for the various aldoses increase in S+H+=SH+ SH+ ---) products

the order, heptoses < disaccharides < hexoses < pentoses, usually proceed more rapidly in DzO than in HgO; DzO is less paralleling the stability order of these compounds and their basic than Hz0 and therefore the substrate (S) competes with

TABLE IV

Relationship of ring conformation and instability factors to mutarotation coeficient, amount of free aldehyde at equilibrium, and substrate eflectiveness to mutarotase

Substrate

Heptose D-Clycero-D-galacto-

heptopyranose

Disaccharides Cellobiose Lactose Maltose

Hexoses D-Glucose D-Galactose D-Mannose D-Culosei

D-Talose

Pentoses D-Xylose D-Arabinose D-Ribose D-Lyxose

M c [utarotatior :oeficienta

0.62 Complex 0.23

0.73 Simple 0.26 0.75 Simple 0.32 0.85 Simple 0.07

1.00 1.27 2.74 3.02

1.00 1.00 1.00 3.4 2.9 0.82 2.7 2.5 0.0

4.15

Simple Complex Simple Simple

Complex

3.21 4.75 7.76 8.98

Simple Complex

7.1 11.7

0.11 0.26

Anomalous 354 Simple 16.7

2.7 6.2

46.1 6.2

Type of mutarotationb

-

4mount of aldehyd@

T-

1 ,

-

-

Bulk rate :onstantd

-

E vfutarotase activity’

lonformation of pyranosef

C-l

C-l C-l C-l

C-l C-l C-l

C-l + 1-c

C-l = 1-c

C-l C-l C-l

C-l * 1-c

Instability factor@

174

1 1 lh

1

174 1,2 1,3,4 (C-l) A2,5 (1-C) 1,2,4 (C-l) H,3,5 (1-C)

1 4

I,3 1,2 (C-l) 3,4 (1-C)

P

4

4 A2 3,4 (C-l) H,1,2,5 (1-C) A2,4 (C-l) H,1,3,5 (1-C)

1,4 3 A2 (c-l) 1,3,4 (1-C)

a These coefficients are for the slow reaction only in cases of complex mutarotation. The values are calculated from those quoted by Bates et al. (5) for reactions at 20” relative to glucose = 1. For a-D-glucose, k = 0.00632 (decimal logarithms, min-I).

6 Complex refers to a mutarotation showing a fast and slow reaction similar to that of galactose. c Results of Cantor and Peniston (23) for 0.25 M solutions at pH 7.0 and 25”, relative to glucose = 1. For glucose, the 70 of aldehyde

(reducible form) quoted by these authors is 0.024 under the above conditions. See also Los et al. (20). d Results of Overend et al. (21) relative to glucose = 1. For glucose, kr = 13 (set-I). e Results of Levy and Cook (16) and Bentley and Bhate (4) relative to glucose = 1. f For D-glycero-D-galactoheptopyranose, the conformation is that predicted on the basis of the known instability factors. For the

disaccharides, the conformation quoted is that for the reducing glucose unit as determined by Bentley (24). All other conformations are those given by Reeves (22).

Q The numbers refer to an axial group other than hydrogen on the carbon atom of that number. The symbol A2 refers to an espe-

cially important condition where the C-O bond of an axial OH group at Cs bisects the two C-O valences of Cl. H indicates the Hassel-Ottar effect when an axial group on Cs occurs on the same side of the ring as another axial group (see Reeves (22)).

h Maltose has an over-all decreased stability compared to cellobiose as a result of boat conformations present in the nonreducing glucose unit (see Bentley (24)).

j The coefficient for gulose refers to that of the calcium chloride complex.

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the solvent for D+ more effectively in DzO than for H+ in Hz0 (12). The mutarotation of glucose was however one of the first examples known of an acid catalyzed reaction proceeding more slowly in D20. Since the observation of k&n > 1 has been interpreted to eliminate a pre-equilibrium proton trans- fer mechanism, a one step slow protonation with simultaneous ring opening (see Fig. 6) was suggested by Bonhoeffer (25) and Bell (26) and more recently was supported by Purlee (27). On the other hand, Challis et al. (28) have interpreted the ob- served ratio somewhat differently. They suggest that a pre- equilibrium proton transfer may occur, leading to a greater concentration of conjugate acid in DzO (see Fig. 7) ; other things being equal, the lcn/kn ratio would therefore be less than 1. But since the hydrogens of the glucose hydroxyl groups equi- librate instantaneously with solvent DzO, bonds broken in the subsequent slow step are C-O and O-H in Hz0 and C-O and O-D in DgO. In this slow rate-determining step, the probable k&n ratio of about 3 for breakage of the O-H bond would overcome the effect of the pre-equilibrium proton transfer, and the over-all process would be slower in DzO.

Although a general agreement as to which mechanism operates in the case of general acid-base catalysis has apparently not been reached, certain conclusions about the mutarotase reac- tion are possible. In all mutarotations so far studied, the k&n ratio for glucose is greater than 1, but the precise value de- pends upon the nature of the catalyst. In the acid-catalyzed reaction, the ratio decreases with increasing acid strength as follows; water alone, 3.8 or 3.16; acetic acid, 2.6; strong acid, 1.37 (29); with tetramethylglucose the ratios are 3.2 for water alone and 1.31 for strong acid (28). In the base-catalyzed re- action, values of 2.35, 2.42 and 2.54 are obtained with chloro- acetate, acetate and trimethylacetate, respectively (30). For the mutarotase reaction, the k&n ratios determined in the present work under a variety of conditions have been within the range 1.73 to 2.12 with glucose-l-H; for one determination with glu- cose-l-D, the ratio was 1.80, and two determinations for the slow mutarotation of galactose-1-H gave values of 1.12 and 1.47. These values are therefore within the range of those found for catalysis by water and acids or bases. It is signif- icant that the kE/kn ratio does not fall below 1, nor does it approach a value of =7 which would indicate a C-H cleavage. The observed ratios are therefore interpreted to indicate an over-all similarity in the spontaneous, acid-, base-, and enzyme- catalyzed mutarotations. In view of the somewhat acid pH optimum of mutarotase it is possible that mutarotase initially catalyzes a reaction of proton addition (“acid” cat.alysis) rather than of proton abstraction (“base” catalysis).

Although the determination of the k&n ratios for the en- zymatic catalysis of mutarotation establishes a proton trans- ferring function for the enzyme, the precise nature of the active site cannot be elucidated from the present results. In view of the observed effectiveness of histidine as a mutarotase ca- talyst (18) a possible role for histidine at the active site was considered. The k&n ratio for histidine-catalyzed muta- rotation was, however, found to be much higher than that observed with mutarotase, or with other acid catalysts. The possibility exists that a lower ratio might be obtained for a specific histidine unit in a protein chain; there was, however, no significant change in the k~/kn ratio when histidylhistidine was substituted for histidine.

Swain and Brown (11) observed that 2-hydroxypyridine, but

OH

FIG. 6. The one-step mechanism for mutarotation. In this and subsequent figures, mutarotase has been represented as E-H and the substrate is a-glucose drawn in the C-l conformation and omitting substituents at carbon atoms other than Cl. Hydrogen atoms which exchange instantaneously with solvent DzO are fol- lowed by the symbol, (D). Since the k&ko ratio with mutarotase was observed to be independent of the time of equilibration of enzyme with D20, it is concluded that the catalvticallv active proton of the enzyme exchanges instantaneously with Dzb. It is therefore justifiable to write mutarotase in DsO as E-D. The reacting base is written as E-, although other possibilities such as water cannot be excluded.

q ; , ‘fast q- ,

bH(D) OH

FIG. 7. The pre-equilibrium proton transfer mechanism for mutarotation.

neither the 3- nor 4-hydroxy compound, is a powerful bifunc- tional catalyst for mutarotation of tetramethylglucose in ben- zene, functioning by a concerted displacement mechanism. 2- Hydroxypyridine does not alter the mutarotation coefficient of glucose itself in water. Swain and Brown noted that the bifunctional catalysis of 2-hydroxypyridine resembled enzymatic catalysis in a number of respects and it was an attractive pos- sibility that mutarotase behaved as a bifunctional catalyst in formation of glucose aldehyde. An approximate evaluation of k2-hydroxypyridine for the tetramethylglucose mutarotation leads to a value of 1.82 (natural logarithms, min-l) which may be compared with our value of 9.75 x lo4 for mutarotase (4) since glucose and tetramethylglucose have similar mutarotation co- efficients. It is apparent that if mutarotase is a polyfunctional catalyst of this nature, other factors must account for its greater activity. It also seemed possible that 2-hydroxypyridine might complex either with glucose, or with the active site of mutaro- tase, and thus function as a specific enzyme inhibitor. The ex- periments reported here show that this was not so.

Consideration of the bonds undergoing cleavage suggests that the observed kn/kn ratio with mutarotase may, in fact, rule out the concerted displacement mechanism of bifunctional catalysis for formation of the intermediate aldehyde. With the HO- C=N- catalytic group found in 2-hydroxypyridine, the reac- tions involved in HZ0 and DzO are compared in Fig. 8. It will be noted that bonds cleaved in Hz0 are C-O and two O-H, and

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1232 Mutarotase from P. notatum. II Vol. 235, No. 5

FIG. 8. The concerted displacement mechanism for formation of glucose aldehyde. S represents substrate glucose which is subsequently written as the chair conformation, although not in the conventional manner. The representation used here is con- venient to indicate the steric interaction with the catalytic group- ing.

inDt0, C-O and two O-D. Although there may be rate differences in the formation of the initial intermediate complex, it seems most likely that the rate determining reaction in- volving the cleavage of two O-D bonds as against two O-H bonds would lead to a much higher k&n ratio than is actually observed.

With a-r-galactose, we have determined kn/kn ratios of 0.64 for the fast reaction and 1.12 and 1.47 for the slow reaction in experiments with mutarotase. For the water-catalyzed, spon- taneous reaction, the kn/kn ratios were respectively 3.76 and 3.10 for the fast and slow mutarotations. Previously, Nicolle and Weisbuch (31), using the Hg line at 5461 rnk reported the ka/kD ratio for the spontaneous mutarotation as 3.3 (16.5’), 3.0 (19”), 3.2 (19”) with cY-n-galactose, and as 3.8 (20”) with P-D-

galactose. They do not state however, whether their constants refer to fast or slow mutarotations, or are an average value. There are apparently no determinations of kn/kn ratios for ga- lactose with catalysts other than water, but it is apparent that there is the same trend from a value of about 3.0 (water) to about 1.0 (mutarotase) as is observed with glucose. It should be noted that the measurements of the fast reaction under our conditions are less precise than for the slow reaction, and there is probably a considerable error in the observed k&n ratio for the enzyme catalyzed rapid reaction. It is also to be noted that the signifi- cance of the rapid reaction with galactose and other sugars is not clear. Although Isbell and Pigman (15) attribute the phenom- enon to a pyranose-furanose interconversion, Rundle and Hen- dricks (32) have shown that their results can be equally inter- preted in terms of the Lowry-Smith mechanism which considers only the three components, o( and /3 anomers and the straight chain aldehyde. A possible confirmation of this conclusion is our observation that although mutarotase catalyzes both reac- tions with galactose, Levy and Cook (16) have shown that the pyranose-furanose transformation in fructose is not catalyzed by mutarotase.

SUMMARY

1. In the mutarotase reaction, there is no introduction of car- bon bound deuterium from solvent DzO and no labilization of the hydroxyl group at C1 of glucose. Substitution of Cr-H by Cr-D has no effect on the rates of spontaneous and enzyme- catalyzed mutarotation. An improved synthesis of glucose-l-D is described.

2. 2-Hydroxypyridine, a bifunctional catalyst for mutarota- tion of tetramethylglucose in benzene, does not inhibit mutaro- tase.

3. For glucose-l-H under a variety of conditions in unbuffered solutions, kn/kn for mutarotase catalysis was within the range 1.73 to 2.12; one determination with glucose-l-D gave a ratio of 1.80 and two determinations for the slow mutarotation of galac- tose gave ratios of 1.12 and 1.47. In 0.005 M phosphate buffer solutions at pH = 6.1 and pD = 6.3 (both being within the pH or pD optimum range) the mutarotase-catalyzed reaction gave kn/kn = 1.78. Histidine, a possible component of the active site of mutarotase, gave a kn/kn ratio with glucose of 3.68; with histidylhistidine, the ratio was 3.85.

4. The general interrelationships between ring conformation and stability on the one hand, and mutarotation coefficient, amount of reducible form at equilibrium and substrate effective- ness to mutarotase, on the other hand, are considered for hep- toses, disaccharides, hexoses, and pentoses.

The reaction mechanism of mutarotase does not involve dehy- dration, any dehydrogenation reaction on carbon-bound hydro- gen, or the hydrated derivative of glucose aldehyde; nor is a single displacement mechanism involved. The enzyme-cata- lyzed mutarotation is essentially similar to the spontaneous or acid catalyzed process; mutarotase is an enzyme with a proton- transferring function.

1.

2.

BENTLEY, R., AND BHATE, D. S., Federation Proc., 18, 190 (1959).

KOSHLAND, D. E., in W. D. MCELROY AND B. GLASS (Editors), The mechanism of enzyme action, Johns Hopkins Press, Bal- timore, 1954, p. 608.

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DIXON, M., AND WEBB, E. C., Enzymes, Academic Press, Inc., New York, 1958, p. 227.

BENTLEY, R., AND BHATE, D. S., J. Biol. Chem., 236, 1219-1224 (1960).

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TOPPER, Y. J., AND STETTEN, D., JR., J. Biol. Chem., 189, 191 (1951).

LEVY, H. R., LOEWUS, F. A., AND VENNESLAND, B., J. Biol. Chem., 222, 685 (1956).

FRUSH, H. L., AND ISBELL, H. S., J. Research Natl. Bur. Stand- ards, 64, 267 (1955).

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SWAIN, C. G., AND BROWN, J. F., J. Am. Chem. SOL, ‘74, 2538 (1952).

WIBERG, K. B., Chem. Revs., 66, 713 (1955). ISBELL, H. S., AND PIGMAN, W. W., J. Research Natl. Bur.

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Ronald Bentley and D. S. BhateMUTAROTATION REACTION

: II. THE MECHANISM OF THEPenicillium notatumMutarotase from

1960, 235:1225-1233.J. Biol. Chem. 

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