THBJODRNAL OF BIOLOGICAL CHEMI~TBY Vol. 244, No. 4,Issue of February 25,~~. 781-788.1969 P~itied in U.S.A.

Studies
III.

on Mutarotases
AND CHARACTERIZATION OF A MUTAROTASE FROM BOVINE KIDNEY CORTEX (Received for publication, July 29, 1968) J. M. BAILEY, PETER H. FISHMAN, From the George Washington University
AND

ISOLATION

P. G. PENTCHEV Washington, D. C. WOO05

School of Medicine,

SUMMARY Mutarotase (aldose I-epimerase) was isolated from bovine kidney cortex in a IO-stage procedure with conventional techniques. The tial product which appears homogeneous in gel diffusion patterns and in sedimentation patterns on sucrose density gradients has a molecular weight of approximately 40,000. Turnover numbers and R, values for the five substrate sugars were D-glucose (1.1 X lo6 min, 25.2 m&r), D-galactose (1.3 X lo6 mm-, 6.5 mu), D-xylose (1.3 X lo6 m&l, 13.2 ~IVI), L-arabinose (2.0 X lo6 min-l, 8.3 mM), and n-fucose (0.35 X lo6 mm-l, 2.0 1~1~). Dialysis against water inactivated the enzyme while lo+ M NaCl restored 50% activity. The preparation is completely stable at 4 but loses activity when stored frozen. It is reversibly inactivated by traces of organic solvents. The enzyme is sulfhydryl-dependent and was completely inactivated by mercuric ions or p-chloromercuribenzoate. The sulfhydryl content measured by titration with p-chloromercuribenzoate was 8 moles of cysteine per mole of enzyme and preliminary amino acid analyses indicated the presence of approximately 18 moles of histidine. Structural requirements for substrates are a pyranose ring with free hydroxyl groups on C-2 and C-3 in the equatorial-equatorial (trans) configuration. Many sugars, including enantiomorphs of the substrates, are good competitive inhibitors. The steric requirements indicate that a three-point attachment to the enzyme is required for substrate activity but that a two-point attachment at C-2 or C-3 or the ring oxygen (or both) is sufficient for inhibition.

Extracts from a wide variety of biological sources have been shown to increase the mutarotation rate of D-ghlCOSe, n-galactose, and several other structurally related aldoses (l-3). The enzymatic nature of the agent responsible for this accelerated mutarotation was first shown by Keilin and Hartree, who consequently named the enzyme mutarotase (1). It was shown that sugars related configurationally to Dglucose (n-xylose and n-arabinose) were substrates for the

bacterial enzyme. Kestonl showed a similar enzyme in mammalian tissues (brain, kidney, lens, liver, and intestine). It was shown that the substrate specificity (n-glucose, n-xylose, Dgalactose, and n-arabinose) was similar to that for the bacterial enzyme although a closer examination of the relative rates with the different sugars suggested that the specificities of the mammalian and bacterial enzymes were not identical. Isolation and partial purification of this enzyme have since been reported from by Bentley and Bhate (4, 5), Escherichia coli Pencillum datum by Wallenfelsand Herrman (6), hog kidney and lensby Keston (7), Li (8), and Lapidesand Chase(9), and greenpepper (10). A comparisonof the relative substrate specificitiesof the enzyme from these diverse sources has been given in a previous paper (10) and confirms the marked similarity of the enzymes. (All catalyze the mutarotation of the samefour substrates,Dglucose,n-galactose,n-arabinose,and n-xylose.) The relative velocities of catalyzed mutarotation of these four sugarsare, however, different with enzymefrom the various sources. A possible participation of the enzyme in sugartransport was proposedby Keston in 1954,l who observed that the highest levels of the enzyme in mammals were found in the renal cortex n-galactose, and that actively absorbed sugars suchasD-&COSe, for and D-XylOSe were substrates the enzyme. A numberof nonmutarotable sugarssuchasar-methyl-n-glucoside I-deoxy-nand glucoseare competitive inhibitors (11). Phloridzin is a potent inhibitor of kidney mutarotaseat concentrationsthat effectively block tubular reabsorption of glucose. This compoundhas no effect on the mutarotase from green peppers. A number of estrogenic agentsfound to block sugartransport in red cellsalso inhibit the kidney enzyme (12, 13). In perinatal rat intestine and kidney, the development of sugar transport mechanismsand mutarotase levels closely parallel each other (14). It has also been claimed by Keston that the kinetic propertiesof hamsterintestine and rabbit kidney mutarotase appear to resembleclosely the kinetics of sugar transport processes thesetissues in (3). It wasshownby Crane, however, that this resemblance often moreapparent than real, is and that KM values calculated for transport of glucoseand certain other sugarsby intestine often differ more than 5-fold from the KM values reported for the correspondingmutarotase (14). Furthermore, we have shown in a previous report (15)
1 KESTON,

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A. S., Fed. Proc., 14, 234(1956).

781

782

Studies on Mutarotases.

III

Vol. 244, No. 4

that the enzyme does not appear to be localized in the brush border region of intestinal mucosa but is found in a supernatant fraction following isolation of the brush border from rat intestine. It was shown in the previous paper in this series (16) that it is unlikely that the enzyme functions merely in some phase of glucose metabolism. None of the common phosphorylating and dephosphorylating systems for glucose display any significant anomeric specificity, and the mutarotational half-life of glucose 6-phosphate under physiological conditions was only 1.5 sec. It was concluded therefore that it is unlikely that rate-limiting anomerizations play any part in glucose metabolism. In this paper the isolation of the enzyme in homogeneous form from bovine kidney cortex is described. A comprehensive study of the structural relationships which determine the substrate and inhibitor speci6city of the enzyme was made and certain physical and chemical properties of the enzyme were examined.
EXPERIMENTAL PROCEDURE

n-Glucose, n-xylose, and n-sucrose were obtained from Fisher. 3-O-Methyl-n-glucose was supplied by Calbiochem and n-allose and n-talose were gifts from Dr. H. S. Isbell, National Bureau of Standards. All other sugars were from Nutritional Biochemicals. Hydroxylapatite (Bio-Gel HT), DEAE-cellulose (Cellex-D), and Bio-Gel P-100 were obtained from Bio-Rad. Sephadex G150 was supplied by Pharmacia. Polarimetric measurements were made with the standard model Keston photoelectric polarimeter unit attached to a Beckman model DU spectrophotometer and fitted with a lo-cm water-jacketed cell which was connected to a thermostated and refrigerated water bath. The instrument was calibrated with sucrose solutions of known optical rotation so that measured optical density readings could be converted to optical rotation. For routine assays, finely powdered a+n-glucose (36 mg) was dissolved in an aliquot of enzyme which had been diluted to 12 ml with EDTA buffer (5 mu, pH 7.4). The optical density of the solution was read at minute intervals. The rate constants for the mutarotation of the sugars were obtained from the equation

where K is the rate constant and cro, at, and (Ye are the measured rotations at zero time, time t, and equilibrium, respectively.
TABLE

The corresponding logarithmic function for each rotation was plotted as the ordinate against time. The slope of such a line represented the rate constant and was expressed in reciprocal minutes. The difference (AK) in the rate constants in the presence (K,,J and absence (Kap) of enzyme is the measure of mutarotase activity. The defined mutarotase unit is that amount of enzyme which at pH 7.4,25, and optimum substrate concentration will catalyze the conversion of 1 pmole of (Y-Dglucose to /3-n-glucose per min. As shown previously (lo), the unit corresponds to a AK of 0.0031 mind1 in the assay system described here. Enzyme PuriJication Procedure-Fresh bovine kidney cortex (1.2 kg) was homogenized in 100-g portions each with 500 ml of 5 mM EDTA buffer, pH 7.4, in a Waring Blendor for 1 min at 2. The crude homogenate was centrifuged at 16,000 X g for 15 min in a Sorvall refrigerated centrifuge. Ammonium sulfate (1.6 kg) was added to 6 liters of the clear supernatant. The ice-cold suspension was sedimented at 16,000 x g for 15 min and the supernatant was subsequently fractionated with an additional 812 g of ammonium sulfate. Following centrifugation at 16,000 x g, the precipitate was dissolved in sufficient phosphate buffer (1 mM, pH 6.4) to give a uniformly dispersed suspension which was then dialyzed against 150 volumes of the same phosphate buffer six times during a 4%hour period. This concentrated dialyzed protein solution was added to a hydroxylapatite column (3.8 x 100 cm) in phosphate buffer (1 mu, pH 6.4). The column was eluted stepwise with 2.2 liters of 50 mu and 2.0 liters of 500 mM phosphate buffer, pH 6.4. Effluent fractions containing the highest mutarotase activity were pooled and the enzyme was subsequently concentrated by the addition of ammonium sulfate to 95% saturation. The suspension was centrifuged at 16,000 x g and the precipitate was dissolved in minimal volume of 1 mu phosphate buffer, pH 6.4. This solution was then added to a Bio-Gel P-100 column (2.8 X 105 cm). The column was filtered with phosphate buffer (1 InM, pH 6.4). Peak enzyme fractions were concentrated as before by ammonium sulfate precipitation and the redissolved protein solution was added to a Sephadex column (2.0 x 80 cm). The column was filtered with phosphate buffer (1 mM, pH 7.4). The effluent fractions which contained the most enzyme were concentrated again by the addition of ammonium sulfate to 95% saturation. The protein precipitate in this case was redissolved in a minimal volume of phosphate buffer (1 mu, pH 7.6) and dialyzed against four changes of 400 volumes
I

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Purification

of mutarotase

from bovine

kidney

cortex
of 5 rnM columns

A total of 1.2 kg of thinly sliced cortex from fresh bovine kidney was used to prepare the original homogenate in 6 liters EDTA buffer, pH 7.4. The table summarizes the major purification steps which are described in the text. Chromatographic were eluted in a stepwise fashion and enzyme and protein were monitored as described in the text.

Fraction

Total

emyme

Total

protein

Specific activity

Fold

purified

RtXXW~ (over-all)

Recovery preceding %

from step

-units Initial 20y0 homogenate. .................. 3%60?& (NH,)zSOh. ........................ Dialysis .................................. Hydroxylapatite (peak fractions). ......... ............ Bio-Gel P-100 (peak fractions) Sephadex G-150 (peak fractions). .......... 95% (NH&SO4 and dialysis. .............. DEAE-cellulose (peak fractions). ......... 6.6 4.0 3.5 2.3 1.12 6.7 5.7 2.6 x x x X x X x X 10 106 106 106 106 101 106 105 m 1.02 x 2.24 X 1.77 x 9.1 x 2.6 X 70 55 10.4
units/mg

%
2.7 3.0 39 66 149 159 385 60.6 53.0 35.4 17.0 10.1 8.6 3.9

106 lo4 104 102 lo2

65 179 198 2,570 4,300 9,570 10,400 25,000

60 88 67 48 59 85 45

Issue of February

25, 1969

J. M. Bailey, P. H. Fishman, and P. G. Pentchev

783

of the same phosphate buffer over 24 hours at 2. The conconcentrated and dialyzed peak enzyme fractions from the Sephadex filtration were subsequently added to a DEAEcellulose anionic exchange resin column with a bed volume of 2.8
50mM Mutarotase Protein -5OOm activity

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IO

15 EFFLUENT

20 VOLUME,

25 ML x lO-2

30

35

FIG. 1. Chromatography of partially purified bovine kidney mutarotase on hydroxylapatite. Column bed, 3.8 X 100 cm; flow rate, 45 ml per hour; elution, stepwise with 2.2 liters of 50 mu and 2.0 liters of 500 mu phosphate buffer, pH 6.4; load, 3.5 X lo6 units and 16 g of protein in 500 ml of 1 mu phosphate buffer, pH 6.4; recovery, 80% enzyme and 56% protein. Arrows indicate range of fractions which were pooled for further processing. 50mM*500mM-+j 0-o 20 c-o Mutarotase Protein activity

IOmMv

2OmM-

%
* zl F: P E . z > !2 w k z s 0 EFFLUENT 5 15

IO

FICA 3. Disc gel electrophoresis patterns of prot,ein fractions during purification of bovine kidney mutarotase. Protein samples were applied to the standard 7% gel column in a Canalco disc gel electrophoresis apparatus model 6 and run for 2 hours at pH 9.5 in 0.05 M Tris-glycine buffer. The developed gels were stained with analine blue black and were electrolytically destained at low current overnight and stored in dilute acetic acid for photography. Samples in order from the top are: initial homogenate, specific activity-64 i.u. per mg (270 pg of protein); ammonium sulfate precipitation, specific activity-179 i.u. per mg (250 rg); dialysis, specific activity-198 i.u. per mg (240 pg); peak enzyme fractions from hydroxylapatite column, specific activity-2570 i.u. per mg (250 pg); peak enzyme fractions from Sephadex G-150 column, specific activity-9570 i.u. per mg (130 pg); peak enzyme fractions from Bio-Gel P-100 column, specific activity-4300 i.u. per column, mg (200 rg); peak enzyme fractions from DEAE-cellulose specific activity-25,000 i.u. per mg (14 pg); peak enzyme fractions from DEAE-cellulose column, specific activity-25,000 i.u. per mg (28Icg). x 105 cm. This resinhad beenpreviously washed and packed in 1 mu phosphatebuffer, pH 7.7. The column wasthen eluted in stepwise fashionwith four phosphatebuffers (pH 7.7), ranging in concentration from 10 MM to 500 mM. Fractions with the highestspecificactivity werepooledand dialyzed for severaldays against successive changes of EDTA buffer (5 mM, pH 7.4). Elution of mutarotaseactivity from all columnswas carried out at 2. Protein in most instances wasdeterminedby the method of Lowry et al. (17) with crystalline serumbovine albuminasthe standard. Purified mutarotase protein assayedby the biuret procedure(18) with the samealbumin standardgave essentially the samevalues asthe method of Lowry et al.

VOLUME,

ML

x IO-

FIG. 2. Chromatography of partially purified bovine kidney mutarotase on DEAE-cellulose. Column, DEAE-cellulose (Cellex-D) with exchange capacity of 0.99 meq per g; bed size, 2.8 X 110 cm; flow rate, 60 ml per hour; elution, stepwise with 700 ml of 1mM,700mlof10mM,1400mlof20m~,600mlof5mM,and1000 ml of 500 mu phosphate buffer, pH 7.7; load, 5.7 X lo5 units of 55 mg of protein in 10 ml of 1 mu phosphat.e, pH 7.7; recovery, 33% enzyme and 64% protein. . ATTOWS indicate range of fractions _. _ _-_ _ which were pooled for further processing.

784
RESULTS

Studies on Mutarotases.

III

Vol. 244, No. 4

Preliminary studies showed that bovine kidney cortex would serve as the best source of the enzyme for large scale purificatim -since this -tissue. bad the. b.i&est m~u~arotase. lee&. based -on. both wet weight and specific activity of soluble protein. The purification is summarized in Table I. The pooling of effluent fractions for subsequent chromatographic separations was a compromise between purity and recovery and is indicated by arrows in Figs. 1 and 2. The final 260,000 units of purified enzyme represented a 3.9% over-all recovery and a 385-fold purification. The degree of homogeneity of the final product and of all of t,he &ages in_thepur&cationprocedure can be judged from the disc gel electrophoretic patterns of the proteins in each preparation (Fig. 3). The final preparation gave a single band on disc gel electrophoresis, and sedimented as a single zone on sucrose density gradients in which the protein distribution and the enzyme activity were completely superimposed. Physical Properties of Enzyme-From a comparison of the migration of mutarotase against standard proteins in a sucrose density gradient, an approximate molecular weight of 40,000 was calculated (Table II). The turnover number of the purified enzyme which had a specific activity of 25,000 Ku. per mg was calculated to be 106 moles of a! to /3-n-glucose per min per mole of enzyme. The enzyme was active over a broad pH range (5.5 to 8.5) with an optimum of 6.5 to 7.5. The optimum temperature fell in the range of 40-43. Precise measurements at this t,emperature could not be made because of the rapid rate of spontaneous mutarotation at the higher temperatures. Activation energies of 22.6 and 11.0 kcal per mole were calculated for the spontaneous and the enzyme-catalyzed mutarotation, respectively (Fig. 4). The activity of bovine kidney mutarotase was significantly influenced by its ionic environment. Exhaustive dialysis against distilled water caused a complete loss of activity which was restored by the addition of NaCl (Fig. 5). Optimum salt concentration was 15 mM, although only 1.5 X 10e6 M NaCl gave 50% reactivation. Similar reactivation effects were observed with KCl, MgCla, and Na2S04. The purified enzyme preparation lost no activity over a 24TABLE

11.0 Keol/Moli~

-3.0

3.2

3.3 I/T

3.4

3.5

X10

on spontaneous and enzymeFIG. 4. Effect of temperature catalyzed mutarotation of ar-n-glucose. The spontaneous (Z&J and mutarotase-catalyzed (AK) mutarotation rate constants of ~-n-glucose were plotted as logarithmic functions against the reciprocal of the absolute temperature. The activation energy was calculated from the slope of this line according to the Arrhenius equation and the optimum temperature from the point at which thelinedeviated from linearity.

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II

OX

I 10-s

I 10-5

I lo-4

I Id3

I lo-2

I lo-

Molecular weight of bovine kidney mutarotase Peroxidase (mol wt 40,000), chymotrypsinogen A (mol wt 25,000), and mutarotase preparations were layered on 5 to 20yo gradients which were subsequently centrifuged with a swinging bucket rotor at 130,000 X 9 for approximately 17 hours. An estimation of the molecular weight of mutarotase was made by comparing the distance traveled from the meniscus by the enzyme and the standards according to the formula (19) D, - = (mol wt of unknown/mol wt of standard)2/3 D, where D, and D, represent the distances migrated by the unknown and standard, respectively.
Molecular Standard protein Trial 1 Trial 2 Trial 3 MealP weight calculated for mutarotase

NaCl CONCENTRATION, MOLES FIG. 5. Stimulation of purified bovine kidney mutarotase by sodium chloride. Purified beef kidney mutarotase, 1 ml, containing 4600 units of enzyme and 180 pg of protein was dialyzed against three changes of 4000 volumes each of distilled water, pH 6.4. Aliquots of the dialyzed solution were subsequently assayed at 25 with 16.67 mu or-n-glucose in 12 ml of distilled water with the addition of increasing amounts of NaCl over a concentration range of 1.5 X 10mBM to 10-l M. Progressive reactivation of the enzyme was obtained which was approximately proportional to the logarithm of the NaCl concentration. In a second experiment, 15 mM solutions of NaCl, KCl, Na2SOd, and MgClz were added to the assay mixture; 100% enzyme activity was restored by the former three salts and 70% activity by MgC12. month period at 4 in 5 MM EDTA buffer. When stored frozen at -2O, however, the preparation had lost 50% of the activity by 3 months and was completely inactive at 8 months. The enzyme was also inactivated by traces of organic solvents (chloroform, carbon tetracbloride, isopropyl ether, and caprylic alcohol).

Chymotrypsinogen A (25,000). 44,500 Peroxidase (40,000) . . . 36,200 a The mean of all trials was 40,500.

43,800 43,200

35,000

44,100 38,100

Issue of February

25, 1969

J. M. Bailey, P. H. Fishman,

and P. G. Pe-ntchev
TABLE

785
III
on

Full activity was restored when the solvents were dialyzed from the preparation. Mercuric compounds were potent inhibitors of the purified enzyme. Full enzyme activity was restored by the addition of n-cysteine. The enzyme activity of known molar concentrations of mutarotase was titrated against mercuric chloride to 100% inhibition. In addition, a spectrophotometric titration with p-chloromercuribenzoate was performed~accordingto-ihemethod of Boyer (20). Known molar concentrations of p-chloromercuribenzoate were added to a fixed amount of enzyme, and the changes in optical density at 250 rnp and in enzyme activity were measured. As shown in Fig. 6, there was a linear relationship between the amount of p-chloromercuribenzoate-sulfhydryl complex formed and the decrease in mutarotase activity. The number of -SH groups per mole of enzyme was calculated to be approximately 10 from the mercuric chloride data and eight from the more precise titration with p-chloromercuribenzoate. In addition, the form of the titration data indicates that all of the sulfhydryl groups are equivalent in reactivity. CatalyticProperties---The effect of mutarotase on the mutarotation of a number of sugars which were available in one or the other anomeric forms, or which could be crystallized in a form containing a preponderance of one anomer, was studied (Table III). Of the 18 sugars tested, the mutarotation rate of only five was increased significantly following addition of the enzyme.

Influence

of bovine

kidney

mutarotase

mutarotation

of sugars

Mutarotation of 0.3y0 solutions of the freshly dissolved sugars in 12 ml of 5 mM EDTA, pH 7.4, was followed with the Keston polarimeter. Rate constants were obtained from the slope of the line plot of log,,, against time where OIL and aB represent the rotations at time t and equilibrium, respectively. Significant differences in mutarotation rates (AK) in the presence (K,,) and absence (K,) of 100 units of enzyme were expressed as the ratio of less than 5yo in these rate constants were K/K,,. Differences not considered significant. Of the 18 sugars listed, nine have been tested previously as substrates for other mammalian mutarotases with essentially the same pattern as described here. o-Galactose, n-glucose, n-xylose, and n-arabinose are substrates for rat kidney (11) and bovine lens (7) mutarotase. n-Arabinose, n-fructose, and n-xylose were tested and found not to be substrates for the bovine lens enzyme and n-rhamnose, n-arabinose, and n-mannose were tested and found not to be substrates for the rat kidney enzyme (7,ll).
Mutarotation rate (K)

Sugar -n-Arabinose ............. n-Arabinose ............. n-Fucose. ............... n-Fucose ................ n-Galactose ............. n-Glucose. .............. n-Glucose ............... n-Glucosamine ......... 2-Deoxy-n-glucose ....... 3-O-Methyl-n-glucose .... D-Lyxose. ............... n-Mannose. ............. L-Mannose. ............. n-Rhamnose ............. n-Talose ................
D-XylOSe ................

Spontaneous 0.138 0.132 0.095 0.098 0.042 0.028 0.028 0.117 0.176 0.026 0.470 0.365 0.324 0.262 0.200 0.094 0.092 0.263
Plus enzyme

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n-Xylose n-Ribose.

................ ...............

0.136 0.900 0.185 0.090 0.640 0.345 0.028 0.117 0.181 0.027 0.445 0.345 0.322 0.275 0.201 0.540 0.088 0.267

0 5.8 0.95 0 14.3 11.3 0 0 0 0 0 0 0 0 0 4.7 0 0

MILLIMICROMOLES

OF

PCMB~ADDED

6. p-Chloromercuribeneoate titration of mutarotase acand sulfhydryl groups. Successive increments (10 pl) of 120 PM p-chloromercuribenzoate (PCMB) in 5 mM Tris buffer, pH 7.0, were added to 560 pl of purified bovine kidney mutarotase (specific activity 25,660 units per mg with a calculated molarity of 1.57 X 10-S) also in Tris buffer and 500 pl of Tris buffer. The
FIG.

tivity

change in optical density at 250 rnp was measured against a blank with the microcuvettes of a Zeiss PM& II spectrophotometer. Ten microliters were removed from both cuvettes after each addition and the enzyme sample was assayed polarimetrically for remaining activity. The results have been corrected for volume changes before plotting. In a similar assay, 35 units of purified bovine kidney mutarotase were incubated with 0.6, 1.2, 1.8, and 2.4 X lo-lo moles of HgClz in 12 ml of 1 nm Tris buffer, pH 7.0, and subsequently assayed for remaining activity with 16.67 rnM a-n-glucose. This residual activity was plotted against moles of HgCls present and extrapolated to 100% inhibition. n-Cysteine, 1.2 X 10-4 moles, was added to an incubation containing HgClz sufficient to give 100% inhibition. The solution was incubated at 25 for 10 min and then assayed for mutarotase activity. Complete activit,y was restored by this treatment. Both titrations gave approximately the same number of --SH groups per mole of enzyme (see text).

These five substrates were n-galactose, n-arabinose, n-xylose, n-glucose, and n-fucose. The kinetics of these substrate-enzyme interactions was studied in some detail. The KM values for Dgalactose, n-arabinose, n-xylose, n-glucose, and n-fucose were 6.5, 8.3, 13.2, 25.2, and 2.0 DIM, respectively (Table IV). The turnover number with all substrates was of the order of 106 moles of (Y anomer converted to /3 per mole of enzyme; L-arabinose with a value of 2 X lo6 moles min-l was shown to be approximately twice as active as the other three substrates (Table IV). The addition of a second sugar to the assay often markedly reduced the enzyme-catalyzed mutarotation of ac-n-glucose. Of the 50 sugars tested, 30 were inhibitors of the enzyme in varying degree (Table V). The inhibition was measured for each sugar over a range of concentrations, from which it was shown that the interactions in each case followed classical Michaelis kinetics for
competitive inhibition (Fig. 7) and from which the-corresponding

K, values were derived as described in the legend to the figure.

When a sugar which was also a substrate was used as an inhibitor, the K, values as expected were the same as their respective KM values. Many sugars which were not substrates were nevertheless extremely potent inhibitors. The affinity of two of

786

Studies on Mutarotases.

III

Vol. 244, No. 4

TABLE IV Michaelis-Menten constants and turnover numbers for bovine kidney mutarotase acting on different substrates Mutarotation rates of freshly prepared sugar solutions were determined in 12 ml of 5 mM EDTA, pH 7.4. The initial velocity of the enzymatic reaction (V), as described earlier, was expressed as aKIIS], where AK1 corresponds to the difference of the forward component

D-Fucose

L-Xylose

2-deoxy-D-Ribose / L-Arabinose / D-Ribose

I
u

I/

Kl b -+P)
of the rate constant in the presence or absence of mutarotase. A linear plot of [S]/V against [S] was used to determine KM. The [S]/V intercept (K&,.,) was used to calculate turnover numbers for each of the substrates by using a molecular weight of 44,600 and a specific activity of 25,000 units per mg for the purified mutarotase.
Substrate

VI

6-

&-Methyl-D-Glucoside

D-Gluronic 4-

Acid

D-Arabinose

Turnover ~0s.~
25.2 6.5

a-n-Glucose.

a-D-Galactose

a-D-Xylose @-I,-Arabinose W.-D- F ucose.

. .

13.2
8.3 2.0

x 106mirr 1.1 1.3 1.3

2.0 0.35

20

4b

6b

80

lb0

Ii0

INHIBITOR
FIG.

CONCENTRATION,

mM

0 Mean of three determinations. a Moles of substrate converted per mole of enzyme per min at 25, pH 7.4, and optimum substrate concentration.
TABLE

7. Competitive inhibitors of purified bovine kidney mutarotase with D-glucose as substrate. The mutarotation rate of 16.67 mM a-D-glucose with 40 units of purified mutarotase was measured in the presence and absencesf varying concentrationsof an equilibrium mixture of a second sugar. The enzyme-inhibitor complex dissociation constants (KI) were obtained from the equation

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V/VI = K,.V
bovine kidney Competitive inhibitors

Km +

bl

KI

[II +1

of

puri$ed

mutarotase

The enzyme-catalyzed mutarotation rate of 16.67 mm cr-D-glucase was determined in the presence and absence of a second sugar added in its anomeric equilibrium form. KI for the sugars was determined by plotting V/VI against [I] where V and VI repreSent the enzymatic velocities in the presence and absence of inhibitor and [I] the concentration of the competitive inhibitor. The intercept of this line with a line representing V/VI = KM/KM + [S] + 1 corresponds to a point on the [I] axis equal to Kr. The following sugars were tested and were not inhibitors (a noninhibitor being defined for practical purposes as a sugar with a KI of greater than 150 mM) : D-arabitol, D-fructose, N-acetyl-D-galactosamine, D-galactosamine, D-galactose g-phosphate, D-glucosamine, N-acetyl-D-glucosamine, D-glucose-6-phosphate, D-hCtOSe, D-mannose, L-mannose, or-methyl-D-mannose, D-raffinose, D-rhamnose, D-sedoheptulose, D-sucrose, D-trehalose, and D-turanose.

where V and VI represent the enzymatic velocities in the presence and absence of the inhibitor (1). When V/V, is plotted against [I], the intercept of this line with a line representing V/V, = (KM/[K~ f S]) + 1 gives a point on the [I] axis equal to KI. That the inhibition shown by this method is competitive in nature has also been shown for a number of sugars both by conventional Lineweaver-Burk plots (21) and by demonstration of reversal of inhibition at higher sugar concentrations (13).

these inhibitors (L-fucoseand L-xylose) was approximately 10 times that of the substrateglucose(Table V).
DISCUSSION

Inhibitor

sugar

.-

KI mdb

Inhibitor

sugar

KI ?nM

D-Fucose............... I,-Fucose............... L-Xylose............... 2%Boxy-wribose D-Galadose D-Allose..

L-Arabinose............ D-Ribose. . a-Methyl-D-glucoside. D-Xylose...............

n-Galacturonic acid..
D-Glucuronic acid. 2-Deoxy-D-glucose.. Galactitol. Erythritol .

2.0 2.6 3.7 5.5 6.3 6.4 8.0 9.8 12.6 14.0 18.4 21.0 24.0 25.4 25.5

L-Arabitol. . 27.7 D-Cellobiose . 31.5 D-Maltose. . . . 32.5 3-O-Methyl-D-glucose. . . 35.5 D-Xylose . . . . 38.0 L-Glucose. . . 44.5 D-Erythrose . . . . . . 47.7 D-Sorbitol . 67.5 D-xy1ito1.. . . . . . . . . 68.5 D-Inositol . . . . . 69.5 D-Arabinose .. . 77 fl-Methyl-D-xyloside.. . 82.5 D-Melibiose.. , 110 2-Deoxy-D-galactose . . . 140 D-Mannitol . . . . . 149

the is In the following discussion assumption madethat sugars that do not competitively inhibit the enzyme-catalyzedmutarotation are also not substrates. From the survey of sugars which were substrates,a pattern of essentialstructural featuresnecessary for substrate interaction with bovine kidney mutarotase was derived. The substitution or removal of the equatorial hydroxyl on carbon 2 abolishesall substrate interaction (2deoxy-D-glucose, D-glucosamine, D-galactosamine, N-acetylD-glucosamine, N-acetyl-D-galactosamine, 2-deoxy-n-galactose). A free hydroxyl on carbon 3 is necessarysince 3-O-methyl-Dglucose not a substrate. An axial-equatorial (cis) relationship is of the hydroxyl groups on carbons2 and 3 abolishes substrate interaction (all mannose sugars,D-talose,and n-lyxose), and the steric relationship at these2 carbonscannot be equatorial-axial (cis) (n-ribose). The required positioning appearsto be equatorial-equatorial @rum) in the proper stereoconfiguration,since all of the substratesdisplayed this characteristic. The steric position of hydroxyl 4 is not critical since D-galactose and Dglucose or D-xylose and L-arabinosewere substrates. A free hydroxyl on carbon5 is not necessary sincethe substrates exist &s pyranose ring structures. The hydroxyl on carbon 6 is not

Issue of February

25, 1969

J. M. Bailey, P. H. Fishman, and P. G. Pentchev

787

decrease in activity may also have been associated with a change in the KM of the enzyme (see below). Similar concentrations (15 mM of KCl, MgC12, and Na$Ok also restored full enzyme activity. Since the reactivation with sodium chloride did not (16). For inhibitor sugars the pattern of essential features is not so display Michaelis type saturation kinetics and because of the simple, and at first sight there appears to be a number of anomrange of salts bringing about reactivation, it seems unlikely that alies. For example, the KM for the substrate n-xylose is 14 mM, any specific cofactor requirement is involved. The concentrawhereas its enantiomorph, n-xylose, is an excellent competitive tion relationship with K+ and Mg+f at the micromolar levels, inhibitor with a K1 of only 3.7 mM. n-Fucose is the substrate of however, has not yet been studied in sufficient detail to rule out highest affinity (KM of 2 mM), yet L-fucose inhibits competitively the possibility that trace amounts of Na+ in these salt preparawith a KI of only 2.6 mM. On the other hand, neither D- nor L- tions are responsible for their activity. mannose displayed any inhibitory capacity. The presence of an The KM value for the purified enzyme acting on glucose, when equatorial hydroxyl group on carbon 2 is essential for a substrate measured in EDTA, Tris, or phosphate buffers with concentrayet, as an inhibitor, 2-deoxy-n-ribose (KI 5.5 mM) has approxitions in the 5 rnM range, lies between 19 and 25 mM. When measmately twice the affinity as n-ribose itself (K I 9.8 mu). Furured in physiological saline, however (Krebs-Ringer bicarbonate thermore, the presence of a pyranose ring is not essential for buffer), the KM of the enzyme acting on glucose decreased to only inhibitory activity since galactitol (K r 25.4 mu), erythritol (K 1 5 mM. It will be noted that this tends to weaken one of the 25.5 IrIM), and L-arabitol (KI 27.7 mM) were inhibitors. On the arguments used by Crane (14) (see above) that the KM for other hand, reduction to the polyol in most cases did markedly mutarotase is much greater than the RM values (1.5 to 2.5 mu) reduce the affinity of a sugar for the enzyme, for example, galacobserved for glucose transport. tose versus galactitol (KI 6.3 mM versus 25.4 mu), n-arabinose The enzyme has an unusually high titratable sulfhydryl conversus L-arabitol (K, 8.0 mM versus 27.7 mu), n-glucose versus tent of 8 moles per mole of protein of molecular weight approxin-sorbitol (KI 25 mu versus67.5 mu), and n-xylose versus D- mately 40,000. Although the nature of the active site has not xylitol (KI 14 mM versus 68.5 MM). None of the keto sugars been further defined, it is possible that histidine is also involved since histidine and peptides containing histidine are good tested had any inhibitory activity. As noted above, substitution of the hydroxyl group at carbon 2 destroyed substrate activity catalysts of mutarotation. It has been suggested by Wallenfels (23) that the catalytic activity may reside in a sulfhydryl group but the substitution allowed retention of inhibitory activity as, for example, with n-glucose versus 2-deoxy-n-glucose (KI 25 IIUVI flanked by 2 histidine residues, and a model catalytic sequence versus 21 mu) and n-galactose versus 2-deoxy-n-galactose (KI involving a concerted proton abstraction and donation in sequence between these groups in the active site and the anomeric 5.5 mu versus 140 MM). Substitution at carbon 2 with an amino or acetylated amino group as in n-glucosamine and n-galactosacarbon atom in the substrate has been formulated. A prelimi_nazy.am.ino. acid analysis. of .the. puri&d enzyme. protein. d.escr.ib.ed . niwz. d 3heic. ~~rivz~,tirres,.c~~e~, .com$etely. destroyedbo.th. substrate and inhibitory capacity. In addition, the presence of here has indicated the presence of 18 histidine residues per mole.2 a phosphate group at carbon 6 as in n-glucose 6-phosphate and This histidine to sulfhydryl ratio of approximately 2, if intern-galactose 6-phosphate completely destroys reactivity both as preted on the basis of Wallenfels suggestion and, taken together a substrate (16) and as an inhibitor. with the extremely high turnover number and the chemical and These diverse requirements for substrate and inhibitor interapparent (but not yet proven) enzymatic equivalence of the sulfhydryl groups, suggests the possibility of multiple catalytic actions with mutarotase appear to involve several unusual situations, particularly as mentioned above, that the enantiomorphs sites on the enzyme molecule.a of the substrates were often excellent competitive inhibitors. REFERENCES We have considered the possibility of alternate inhibitory sites. 1. KEILIN, D., AND HARTREE, E. F., B&hem. J., 60, 341 (1952). However, by taking into account the different factors which 2. LEVY, G. B., AND COOK, E. D., Biochem. J., 67, 59 (1954). affect the conformational status of the sugar ring (22), it has been 3. KESTON, A. S., J. Biol. Chem., 239, 3241 (1964). found possible to define a single set of common structural features 4. BENTLEY, R., AND BHATE, D. S., J. Biol. Chem., 236, 1219 which rationalizes most of the experimental findings without the (1960). 5. BENTLEY, R., AND BHATE, D. S., J. Biol. Chem., 236, 1225 necessity of proposing two separate binding sites for the sugars. (1960). The turnover number of the enzyme (between 1 and 2 x lo6 6. WALLENFELS, K., AND HERRMAN, K., Biochem. Z., 343, 294 min-r depending on the substrate) is among the highest recorded (1965). for an enzyme and accounts partly for the extremely high activity 7. KESTON, A. S., Arch. Biochem. Biophys., 102, 306 (1963). 8. LI, L. K., Arch. Biochem. Biophys., 110, 156 (1965). found in certain tissues. For example, 1 g of bovine kidney cor0; LAPIDES, S. L., AXD JELUE, P,. N., Biochem. Biophys. Res. tex contains sufficient enzyme under optimum conditions to conCommun., 31, 967 (1968). vert approximately 2 g of c~ to P-n-glucose per min. From the 10. BAILEY, J.M., FISHIMAN, P.H., AND PENTCHEV, P. G., J. Biol. specific activity of the preparation described here, this correChem., 242, 4263 (1967). sponds to approximately 2 mg of the pure enzyme protein per g 11. KESTON, A. S., Science, 120, 355 (1954). 12. LEFEVRE, P. G., Pharmacol. Rev., 13, 39 (1961). of kidney- tissue. 13. BAILEY, J. M., AND PEN,TCEZ~, 1; G., A,wwr.:.J. Phyriot., 208; The catalytic activity of the enzyme was completely lost 385 (1965). 14. CRANE, R. K., Handbook of physiology, Academic Press, New following dialysis against distilled water. This activity was reYork, 1967. p. 1340. stored by the addition of small amounts of sodium chloride to the assay system, only 1.5 pM serving to restore 50% of the activity. The optimum salt concentration was 15 mM and physiological salt concentrations caused some decrease in activity (Fig. 4). This 2 J. M. Bailey and P. H. Fishman, unpublished data. ~FIs~~AN, P. H., BAILEY, J. M., AND PENTCHEV, P. G., Fed. Proc., 26, 655 (1966).

critical since n-fucose, 6-deoxy-n-galactose, and the pentoses Dxylose and n-arabinose are substrates. Substitution at carbon 6 with a phosphate group, however, destroys substrate capacity

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15. BAILEY, Med., 16. BAILEY, Biol. 17. LOWRY, R. J., 18. GORNALL, Biol. 19. MARTIN,

Studies on Mutarotases.
J. M., AND PENTCHEV, P. G., Proc. ,540~. Exp. Biol. 116, 796 (1964). J. M., FISHMAN, P. H., AND PENTCHEV, P. G., J. Chem., 243, 4827 (1968). 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, J. Biol. Chem., 193, 265 (1951). A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Chem., 177, 751 (1949). G., AND AMES, B., J. Biol. Chem., 236, 1372 (1961).

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20. BOYER, P. D., J. Amer. Chem. Sot., 76, 4331 (1954); in J. Q. WEBB (Editor), Enzyme and metabolic inhibitors, Vol. II, Academic Press, New York, 1966, pp. 763-76G. 21. BAILEY, J. M., AND PENTCHEV, P. G., Biochem. Biophys. Res. Commun., 14, 161 (1964). 22. DYKE, S. F., The carbohydrates, Interscience Publishers, New York, 1960, p. 62. 23. WALLENFELS, K., HTJCHO, F., AND HERRM~N, K., Biochem. Z., 343, 307 (1965).

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