Kinetics and Mechanism of Action of Glucose Oxidase* · Turnover of Enzyme-The results of manometric and stopped flow experiments are in satisfactory agreement, as illustrated in

THEJOURNAL ~~BIOLOGICALCHEMISTRY Vol. 239, No.11, November 1964

Printed in U.S.A.

Kinetics and Mechanism of Action of Glucose Oxidase*

QUENTIN H. GIBSON, t BENNETT E. I?. SWOBODA,$ AND VINCENT MASSEYI

From the Department of Biochemistry, University of Shefild, Shemld, England

(Received for publication, iLlay 13, 1964)

The glucose oxidase from molds has been st.udied by a number of workers (l-8). The enzyme catalyzes the oxidation of n-glucose to n-glucono-b-lactone (7). Its substrate specificity has been examined, and it also attacks 2-deoxy-n-glucose, n-mannose, n-galactose, and n-xylose (5), although the turnover numbers for the last three sugars are very low.

In this laboratory in recent years the formation of semiqui- nones in the oxidation and reduction of free flavins has been studied by spectrophotometry and electron spin resonance (9, lo), and attempts have been made to apply the results from model systems to flavin enzymes. No general relationship has been found between electron spin resonance signals and the appearance of absorption spectra corresponding to enzyme forms intermediate between fully oxidized and fully reduced flavin, nor do such oxidation-reduction intermediate forms necessarily play a part in catalysis (11). Each enzyme must therefore be examined separately, and the results of electron spin resonance and optical spectrophotometry correlated with rapid reaction studies, to establish whether or not intermediate forms of the enzyme react at rates sufficient to account for its catalytic activity. Electron spin resonance studies of glucose oxidase have already been reported by Beinert and Sands (12) and by Mason et al. (13), and while our own experiments on the glucose oxidase from AspergiZZus niger were in progress, Nakamura and Ogura (14) published an important paper on the kinetics of the oxidation of glucose by the enzyme from Penicillium arnagaskiense. Briefly, these papers indicate that semiquinoid intermediates play no part in the catalytic mechanism of glucose oxidase.

This paper describes kinetic experiments by manometric and stopped flow methods with several substrates. Our experimental results confirm and extend those of Nakamura and Ogura (14), although our additional experiment.s suggest that a different interpretation is required to accommodate all the observations. Whereas Nakamura and Ogura identified a first order rate-limiting step with the conversion of enzyme-glucose complex to reduced enzyme and product, our results can be adequately explained without postulating the formation of an enzyme- glucose complex, and we propose the dissociation of product from an enzyme-product complex as a first order rate-limiting step. With 2-deoxyglucose, however, there seems to be a typi-

* This work has been supported by the United States Public Health Service under Grants RG-8116 and GM 11231.01 and by the United States Air Force under Contract E.O.A.R. 61/i. One of us (B.E.P.S.) is indebted to the Department of Scientific and Industrial Research for a scholarship for training in research methods.

t Present address, Johnson Research Foundation, University of Pennsvlvania, Philadelphia 4, Pennsylvania.

$ Present addiess, Departmend of Biochemistry, University of Michigan, Ann Arbor, Michigan.

cal enzyme-substrate complex, and the rate of dissociation of product does not limit the rate of enzyme turnover.

EXPERIMENTAL PROCEDURE

Materials

Pure glucose oxidase (A. niger) was prepared from Dee-O- Concentrate obtained from Miles Chemical Company by a modification of the method of Kusai (8).

Catalase (beef liver) with 1260 Keilin units per g was obtained from L. Light and Company, Bucks, England.

n-Glucose, n-galactose, D-xylose, n-mannose, and D-glucono-6- lactone were obtained from British Drug Houses, Ltd., Poole, Dorset, England.

2-Deoxy-n-glucose (A grade, glucose-free) was obtained from the California Corporation for Biochemical Research.

Purity of Sugars-The galactose, xylose, and mannose were tested for purity and glucose contamination both chromato- graphically and enzymically.

1. Ascending chromatography on Whatman No. 4 paper in a solvent system consisting of ethyl acetate-pyridine-water (2 : 1: 2, v/v) revealed no glucose contaminations, but a very small contamination of the galactose with lactose. However, glucose contaminations of less than 1% were difficult to detect by chromatography.

2. The rate of glucose oxidation catalyzed by glucose oxidase is very much faster than the oxidation of mannose, galactose, or xylose. Minute glucose contaminations can be detected in manometric assays, since, if suitable concentrations of one of these sugars and enzyme are used, any significant glucose contamination gives rise to a fast initial rate of oxygen uptake, followed by a slower, constant rate of uptake due to the princi- pal sugar alone. At 0.67 M each, mannose, galactose, and xylose were found to be contaminated by glucose approximately 0.2, 0.2, and 0.1 ‘%, respectively. (One batch of galactose had a 2% contamination with glucose and was rejected.) The glucono-d- lactone was found to have approximately 0.1 $ZO contamination with glucose.

Glucose-free Mannose, Galactose, and Xylose-Glucose-free sugars are required for anaerobic stopped flow experiments. Glucose contaminations were reduced to an insignificant level enzymically, by incubating the contaminated sugars at 35’ for 1 hour in air (by shaking) with a minute amount of glucose oxidase. The amount of glucose oxidase was calculated to be sufli- cient to oxidize almost all the glucose in this time, but insufficient to convert more than 1% of the sugars to product,s. Enaymic action ceased when the sugar solutions were made anaerobic. In addition, very pure galactose was prepared (15) from penta-

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3928 Kinetics of Glucose Oxidase Vol. 239, No. 11

acetylgalactosel that had been recrystallized a number of times from methanol. The stopped flow results obtained with this pure galactose were in good agreement wit,h those obtained with enzymically treated galactose.

Buffers-In both manometric and stopped flow experiments, 0.1 and 0.13 M sodium phosphate buffer, pH 5.6, were used. Keilin and Hartree used phosphate buffer, pH 5.6, in their experiments with glucose oxidase (5, 6). Manometric experiments have also been performed with 0.1 M sodium acetate buffer, pH 5.6, as used by other workers (8). No difference in results has been obtained with the two buffers, although both are at the ends of their respective buffering ranges.

In experiments with glucono-&lactone, 0.2 M sodium phosphate buffer, pH 6.3, was used.

Concentration Measurements-All sugar solutions made up from solids were allowed to mutarotate to the equilibrium mix- tures of (Y and p forms before use in kinetic experiments. Ki- netic data in this paper are expressed in terms of total sugar concentration, although glucose oxidase is specific for P-glucose.

The enzyme concentration was measured in terms of its flavin content. In stopped flow experiments, solutions of known oxygen content were prepared by equilibration with room air and measured barometric pressure in open vessels, or by equilibration with pure oxygen in glass tonometers. The concentration of oxygen was calculated from tables of its solubility. Be- cause high concentrations of glucose depress the solubility of gases in water, as a general rule the enzyme solution was equili- brated with oxygen, and the glucose solutions were freed from dissolved gases by shaking and evacuation. The tonometers were filled with prepurified nitrogen (British Oxygen Company, Wembley, Middlesex, England), and the oxygen-free solutions were delivered directly into the stopped flow apparatus.

Methods

Manometric Experiments-Standard Warburg manometers were used. The rates of oxygen uptake in the presence of pure glucose oxidase and catalase (100 fig per manometer) were studied over a range of sugar concentrations (0.1 to 0.01 M)

at pH 5.6, at 0” and 25”, and at different oxygen tensions. The gas space of the manometer was filled with either oxygen, air (21% oxygen), or 10.5% oxygen. In general, the amount of enzyme per manometer was adjusted so that oxygen uptakes did not exceed 3 ~1 per minute. The manometers were shaken at a constant rate of 150 per minute. The oxygen uptakes over 25 minutes were used in calculating the rate of oxygen uptake, since the rates were constant for this time.

Contaminating glucose (0.0002 ivr or less) in mannose, galactose, and xylose at concentrations of 0.1 M was used up so rapidly that only the rates in the first 2 to 5 minutes were greater than at subsequent times. In fact, at concentrations of less than 0.1 M sugar, glucose contamination became increasingly difficult to detect at all. However, the constant rates of oxygen uptake at times greater than 5 minutes after mixing were used for calcu- lation purposes.

Stopped Flow Experiments-These were carried out with the apparatus of Gibson and Milnes (16) with the use of a 2-cm light path. A majority of the experiments were performed at 450 rnp with a tungsten light source; for some, in the ultraviolet region, a 150-watt, direct current xenon arc was used. The

1 Sample kindly given to us by Dr. P. Finan, Chemistry De- partment, Sheffield University, England.

modification of the stopped flow apparatus described by Hast- ings and Gibson (17) was used to follow rapid changes in protein fluorescence.

Effect of Diffusion in Manometry-It is obvious that a concentration gradient is required across the gas-liquid interface to permit the uptake of oxygen in a Warburg manometer. In many applications of manometry, this gradient may be disre- garded, but not when the kinetics of an enzyme like the glucose oxidase or n-amino acid oxidase is under study, since the oxygen concentration in the liquid phase is one of the variables in the experiment. The oxygen concentration in the liquid phase is always less than that calculated from the tension in the gas phase and the solubility of oxygen, and may be estimated as suggested by Roughton (18). When a constant rate of oxygen uptake has been reached, it may be shown that

[O,lL = apo2 (VM - V)/VM

where [O& is the concentration of oxygen in solution, cr is the solubility coefficient of oxygen, poZ is the partial pressure of oxygen in the gas phase, V is the rate of the enzyme reaction, and VN is the rate of the enzyme reaction at, infinite enzyme concentration, i.e. when the rate is limited by diffusion only.

This equation has been used to estimate the errors introduced into the manometric experiments by limitation of diffusion. It has been found that with our particular experimental conditions, errors in rate measurements are less than 5% with uptakes of up to 5 ~1 per minute with air in the flasks. With lower oxygen percentages, accurate manometry is scarcely feasible. Cor- rections have been applied to manometric results where appropriate. In no case did these corrections exceed 5% of the amount measured.

RESULTS

Turnover of Enzyme-The results of manometric and stopped flow experiments are in satisfactory agreement, as illustrated in Fig. 1, which shows an experiment with glucose. The manometric results have been calculated with absorbance for the enzyme flavin at 450 rnp of 14.1 X lo3 cm2 per mmole,2 assuming that in the presence of catalase the over-all reaction is

Glucose + 3 02 + gluconic acid + He0

Corrections have been made for limitation by diffusion, where this was significant.

In turnover experiments by the stopped flow method, solutions of oxidized enzyme containing known concentrations of oxygen were mixed with oxygen-free solutions of glucose. An experiment showing the changes in absorbance at 450 rnp is given in Fig. 2. In the example, the change in optical density at 450 rnp corresponding to fully oxidized enzyme would be 0.245. In the “steady state” condition, with an initial concentration of oxygen of 6.8 X 10m4 M, the change in optical density is at first 0.192; as the oxygen is used up by enzyme turnover, the change in optical density falls, at first slowly, and then, when the oxygen is almost exhausted, much more rapidly, as the enzyme is converted progressively into the reduced form. It is necessary either to use enzyme preparations free from catalase or to add cyanide, in order to prevent spurious results due to liberation of O2 from the Hz02 produced in the enzymic reactions.

Evaluation of Stopped Flow Experiments-Turnover numbers

2 B. E. P. Swoboda, unpublished.


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November 1964 Q. H. Gibson, B. E. P. Swoboda, and V. Massey 3929

have been obtained from experiments of the kind shown in Fig. 2 by following the general principles of Chance’s (19) classi- cal paper. I f glucose is in sufficiently great excess relative to oxygen so that its concentration may be treated as constant throughout the reaction, then, provided that glucose reacts with a form of the enzyme having the spectrum of the oxidized enzyme, the turnover number at any instant is proportional to the ordinate in Fig. 2. The total number of times that the enzyme must turn over to exhaust the added oxygen can be calculated from the conditions of the experiment, and this number may be equated with the area enclosed in Fig. 2. The turnover number corresponding to a given value of the ordinate may be found by measuring the proportion of the total area enclosed between two ordinates near the beginning of the curve, where the rate of change of optical density is small. On the assumption that the corresponding amount of oxygen has been consumed in the time interval between the ordinates at a constant rate corresponding to the average of the first and last ordinates, the turnover number corresponding to a particular ordinate may be found.

Turnover Rates with Various Sugars and under Di$erent Con- ditions-The results with glucose at 0” have already been presented in Fig. 1. The substance of a reciprocal plot giving rise to parallel lines like those in Fig. 1 can be summarized in three constants: the rate with infinite concentrations of both substrates (V,,,), and the concentration of each substrate required to give half of this maximum rate in the presence of an infinite concentration of the other (Ko, and Kpiucose). Some values of these quantities are collected together in Table I.

With 2-deoxyglucose, the results are similar in form, but the numerical values of the constants differ widely. With 2- deoxyglucose, the K o2 is so small that it is difficult to determine accurately, while I’,,, is much less than for glucose under comparable conditions.

The sugars mannose, galactose, and xylose are attacked more slowly still. The form of the kinetic results is different in that there is no evidence for the existence of a limiting velocity, even when very high concentrations of these sugars (0.5 M) are used, as has been done in stopped flow experiments. An example is given in Fig. 3. In these cases the kinetic results are fully specified by giving the turnover number at any one sugar concentration. Koz cannot be determined as it is impracticable to work with 02 concentrations sufficiently low to influence the rat,e of reaction.

Effect of Varying Wave Length of Observation-An extensive series of determinations has been made over the range of 250 to 470 rnk with several glucose concentrations. In every case there has been excellent correlation between the results, although at the shortest wave lengths correction is needed for the absorbance of HzOz formed during the reaction.

Observation of Fluorescence Changes during Turnover-The apparent fluorescent yield of the aromatic amino acids of glucose oxidase changes on oxidation and reduction, and this change has also been used to follow the reaction, exciting at 280 rnw and observing the fluorescence through a combination of 3 mm of Lucite and 1 mm of OX7.3 The changes in fluorescence correlated within experimental error with changes in absorbance. One explanation for the change in fluorescence of the aromatic amino acids on oxidation and reduction of the enzyme is that an

3 Corning Glass Works, Corning, New York.

Turnover Experiments.

0.04 -

l/Oxygen (mM)

FIG. 1. The rate of oxidation of glucose by glucose oxidase measured in manometric experiments ( l ) and stopped flow spectrophotometric experiments (0). The numbers by the lines give the concentrations of glucose. Conditions: 0”, 0.13 M phosphate buffer, pH 5.6. In the spectrophotometric experiments, the enzyme concentration was 1.17 X 10V5 M after mixing. The reaction was followed at 450 rnM in a 2-cm light path in the presence of 1 X 10v3 M KCN. In the manometric experiments, the enzyme concentration was 2 X 10e8 M; total volume in the flask was 3 ml, with 100 pg of catalase per flask. Rates were corrected for diffusion as described in text, and allowance was made for the effect of catalase.

1 Stop Flow Record

0 I

0.5

Time (se4

I 1.0

FIG. 2. Reproduction of oscillograph trace obtained with stopped flow apparatus in following the reaction between glucose and oxygen. At the time shown by the arrow, a solution of enzyme in equilibrium with 1 atmosphere of 02 was mixed with an equal volume of &-free 0.1 M glucose. The reaction was followed in a 2-cm light path at 450 rnM, at 0” in 0.13 M phosphate buffer, pH 5.6; the concentration of enzyme was 1.17 X 10m5 M.

alteration in overlap of flavin absorption and amino acid emis- sion takes place, and is associated with a varying degree of energy transfer. The correlation between absorbance and fluorescence changes supports this explanation and argues against an intramolecular change in configuration of the protein molecule.

Half-reaction Experiments-The reaction of the oxidized enzyme with sugar was followed by mixing thoroughly deoxy- genated enzyme with oxygen-free sugar solution and observing the decrease in absorbance at 450 rnp in the stopped flow apparatus. In every case except that of 2-deoxyglucose, the rate of appearance of reduced enzyme flavin was proportional to the concentration of sugar used up to the highest concentrations which can be employed in the stopped flow apparatus. (The upper limit is set by the appearance of spurious changes in


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3930 Vol. 239, No. 11 Kinetics of Glucose Oxidase

TABLE I Kinetic quantities for glucose oxidase

The results quoted are for stopped flow experiments, except for mannose, galactose, xylose, and the 25” experiment with 2-deoxyglucose, in which manometric experiments supplemented stopped flow runs. The results with glucose at 0” are the average of three concordant experiments with different preparations of enzyme. The other results are from single experiments. The schemes to which the rates apply are the following. For glucose, mannose, xylose, and galactose,

For 2-deoxyglucose,

Sugar

Glucose. ................. Glucose .................. Glucose. ................. Glucose ..................

Mannose ................. Galactose ................ Xylose ...................

2-Deoxyglucose. .......... 2-Deoxyglucose. ..........

-

--

-

Temper- ature K,Oz kl ka ka k4 k-~/k~

--

0” 13 27 38

V msx

se?9 M

235 590

1,150 2,000

M vmf M-1 seco

0.12 0.21 2,100 0.11 0.29 5,500 0.11 0.48 10,000 0.12 0.83 16,000

25 22 25 8 25 3

0 25

-~

15 0.025 50 0.04 0.03

-

l/Xylose (M) Half-reaction.

Eox + G L Ew.8 k2 1 ------+ Ered + 6-lactone

E k3 red + 02 - EL, Pz ka E,, + Hz02

kl k2 E,, + D e E,,D -----+ Eled + b-lactone

k-1

Ered + 02 - k4 Em + Hz02

2 4 6 8 I I I I ' /

l/Xylose CM)

FIG. 3. The oxidation of xylose by glucose oxidase measured manometrically. Open points, scales at left and bottom of figure. Conditions: 25”, 0.13 M phosphate buffer, pH 5.6; 0.1 mg of catalase per flask. Filled points show the reduction of glucose oxidase (1.1 X lO+ M) by xylose, measured in a stopped flow apparatus. Scales at right and top of figure. Conditions: 25”, pH 5.6, 2-cm light path, 450 rnp; enzyme and sugar solutions anaerobic.

absorbance due to scattering across refractive index gradients, and is about 0.5 M for sugars such as glucose.) With Z-deoxyglucose, there was a clear maximum velocity and an easily measured half-speed concentration. The results for the different sugars are collected in Table I in the column headed kr and show that as in turnover experiments, glucose reacts more rapidly than the other sugars. The results with 2-deoxyglucose are given in Fig. 4.

Inhibition Experiments-Glucose reacts so much faster than

7

se0

650 3,300

50

M-1 WC-1 se0

1.3 x 106 370 1.7 x 106 720' 2.1 x 106 1,150 2.4 X lo6 2,000

-7

M -

0.04

0’ /

OW 0

11 Z-deoxyglucose(M)

kr

al-1 wx-

1.7 x 106

FIG. 4. The reaction of oxidized glucose oxidase with 2-deoxyglucose measured spectrophotometrically at 25’ in 0.13 M phosphate buffer, pH 5.6, with a stopped flow apparatus. Enzyme concentration after mixing, 1.0 X 1OP M; 2-cm light path, 450 rnp; enzyme and sugar solutions anaerobic.

any other sugar that the other sugars can easily be tested for inhibitory activity. It was found that 2-deoxyglucose is a competitive inhibitor of glucose to the extent predicted from the mechanism of the reaction of these sugars given below (“Discussion”). The results of a manometric experiment are shown in Fig. 5.

Nakamura and Ogura (14) have reported that n-glucono-6- lactone is an inhibitor of glucose oxidation. This has been confirmed in turnover experiments by the stopped flow method. The inhibition is a weak one, however, since with 5 x 10-J M

glucose and 5 X lo+ M glucono-Slactone there was only a 30% effect on the rate. These experiments were done in 0.2 M phosphate buffer, pH 6.3, to minimize possible pH changes pro-


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duced by spontaneous hydrolysis of the lactone. The solutions were used within 2 minutes of preparation. It was found that the distribution of enzyme between the oxidized and reduced forms was unchanged during turnover. This result is considered further below.

DISCUSSION

As a preliminary to consideration of the mechanism of the reaction, it is necessary to decide the number of spectrophotometrically distinguishable forms of the enzyme. It has been found that (a) there is close correlation of changes with time at all wave lengths examined, and (6) no absorbance changes have been seen under any conditions at wave lengths higher than 0 0.02 0.04 006 0.08

the main flavin absorption band at 450 rnp. These results are 2-deoxyglucose (MI

in agreement with the suggestion of Nakamura and Ogura (14) FIG. 5. The effect of varying concentrations of 2-deoxyglucose

that, unlike some other flavin enzymes, the glucose oxidase does on the oxygen uptake of glucose oxidase in the presence of 0.1 M

not operate with a typical semiquinone intermediate form of glucose. Manometric experiment at 25”; 1.2 mM 02; 0.13 M phos-

finite lifetime. This conclusion is further supported by the ex- phate buffer, pH 5.6. The line is calculated from the expression given in the text, by using the rate constants quoted in Table I.

periments with the free semiquinone prepared by chemical ;-

means reported in a previous paper (11). For these reasons, all oxygen is fully accounted for by the simple.‘scheme above with

the kinetic conclusions presented later in this discussion are the additional supposition that the reoxidation of the reduced

based on the suppositions that there are only two spectrophoto- enzyme by molecular oxygen is rapid compared with its reduction

metrically different forms of the enzyme, oxidized and reduced, by 2-deoxyglucose.

and that in catalysis the enzyme alternates between them. How- The half-reaction results with the other sugars are different.

ever as discussed later, we believe that the chemical nature of There is no clear I’,,,, as the reciprocal plots pass through the

the reduced enzyme is not yet established. origin. This result will be found if k-1 is small or zero, and

The manometric and stopped flow turnover experiments kz > kJsugar). (If kz is of the same order as kl[sugar], the time

which yield Lineweaver-Burk plots consisting of a set of parallel course of enzyme reduction will be sigmoid, and the reciprocal

lines, and which give a finite I’,,, on extrapolation to infinite plot nonlinear.) I f kz is taken > kJsugar], for practical pur-

concentrations of both substrates, require a rate equation of the poses this amounts to discarding the species E,,S, since its

form l/v = l/V,,, + A/[oxygen] + B/[sugar], where A and B concentration will always be small. The reaction of oxidized

represent single or grouped constants. A rate equation of this enzyme with sugars other than 2-deoxyglucose may thus be

kind may be obtained from any scheme which includes, as a reduced to

minimum, one first order and two second order steps, with the condition that the two second order steps occur independently (i.e. no kinetically significant ternary complex). All the rate which sufficiently describes the experimental results. This

measurements are fully accounted for when the constants V,,,, raises the interesting question whether glucose, mannose, xylose,

A, and B have been determined. For glucose, all three con- and galactose actually form enzyme-substrate complexes as this stants can be determined from the experimental results; for term is usually understood. It appears from the experimental 2.deoxyglucose, the oxygen reaction is so much faster than the results that if a complex is formed at all, the affinity of the enzyme reaction with sugar that the value of A is uncertain, while for for substrate must be strikingly low, requiring concentrations of

mannose, xylose, and galactose, only B is measurable. the order of at least 5 to 10 M for half-saturation. A more detailed picture of the reaction can be obtained by When the reduced enzyme is prepared by mixing enzyme

considering the reaction of the oxidized enzyme with sugars and under anaerobic conditions with an equivalent amount of glucose

of the reduced enzyme with oxygen studied separately in the and the rate of the reaction with oxygen is measured in the

stopped flow apparatus (i.e. the half-reaction). With 2-deoxy- stopped flow apparatus, there is again no evidence for the for-

glucose, the results may readily be interpreted in terms of the mation of an enzyme-substrate complex, and the reaction with

scheme oxygen may be written

E red + 02 + Eox + HzOz

K-1 Two simple half-reactions of the form

where the subscripts indicate the spectrophotometric state of E,, + 6’1 -+ Ered:Ered + Sz + Em the enzyme. Provided that k-1 >> kz, the reciprocal of the rate constant of the spectrophotometric change plotted against the put together provide a sufficient scheme to account for the

reciprocal of the concentration of sugar will give a straight line kinetics of oxidation of mannose, xylose, and galactose. For

intersecting the ordinate at l/i&. The value of the dissociation 2-deoxyglucose, a slight expansion is required to explain the

constant for the enzyme-substrate complex in the above scheme, finding of a maximum rate, and the scheme becomes

Ic_,/i&, is given by dividing the slope by the intercept and is kz equal with 2-deoxyglucose to the value of K, (0.04 M) found in Eox + 81 + E,,& __f Ered + product

1 cat,alytic experiments. As illustrated in Fig. 4 and Table I, the behavior of the enzyme in turnover with 2-deoxyglucose and

November 1964 Q. H. Gibson, B. E. P. Swoboda, and V. Massey 3931

Ered + 02 k4

- Eox + HzOz


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0.06 -

"0 50 100 150

l/Glucose(M)

FIG. 6. Analysis of the reaction with glucose. Line A is a plot of the rate of the half-reaction, E,, + glucose -+ Ered P, as calculated from turnover experiments by the method described in the text. Line B gives the rate of the reaction as determined by mixing oxidized enzyme with glucose in the stopped flow apparatus. The inset shows the bottom left-hand corner of the main diagram on an enlarged scale. Conditions of the experiment: O”, 0.13 M

phosphate buffer, pH 5.6, 2-cm light path, 450 rnB; enzyme concentration, 1.17 X low6 M after mixing. Experiments were conducted in the presence of 1 X lo+ M KCN.

The case of glucose is more complicated, and requires special treatment. As already pointed out, the manometric turnover experiments require one first order and two second order steps, and although the half-reaction experiments provide numerical values for two second order steps, each of which is associated with a spectrophotometric change in the enzyme flavin, they do not yield values for the first order step. This first order step cannot be associated with a spectrophotometric change. The two sets of experiments, manometric and stopped flow half- reaction experiments, can, however, be satisfied by a scheme such as

E, + Sl ---f Ed’, + Ez + PI:E~ + Sz + El + Pz

where 81 and Sz are the substrates and PI and PZ the products of the two half-reactions, and El and EQ are the two spectrophotometrically distinct forms of the enzyme. The notation used for the forms of the enzyme has been changed from that used earlier because at this point in the argument S1 and SZ are not specifically identified, nor can El and Et be assigned to the oxidized or reduced forms of the enzyme. The observed V,,, is identified with the first order rate-limiting step, written E2P + EQ + PI, although the implication of a product is, of course, arbitrary.

A closer examination of the reaction mechanism leading to more precise specification is possible if the half-reaction results and the spectrophotometric turnover experiments are taken together. In a turnover experiment, the area under a curve such as that of Fig. 2 represents the product (concentration of oxidized enzyme) x (time) required to allow the number of turnovers needed to exhaust the oxygen initially present in the system. The turnover rate at each glucose concentration can at once be calculated for the oxidized fraction of the enzyme separately. This has been done for a series of glucose concentrations in an experiment at O”, and the results are plotted in Fig. 6. The points lie on a straight line (A), which has the same slope as the line (B) representing the stopped flow half-reaction results, but A has an intercept at infinite glucose concentration

corresponding to a rate constant of 370 set-1. This may be interpreted either as the rate at which product (now identified as H202) can dissociate from the oxidized enzyme, leaving enzyme free to react with glucose, or as the rate at which some intramolecular change occurs which must take place before reaction of oxidized enzyme with glucose is possible.

The rate constant of 370 set-l is greater than the limiting rate of 235 set+ turnover at infinite concentration of both substrates. Another first order step must therefore be introduced, so that the complete scheme is

kl E,, + glucose - EredPl kz

- Ered + PI

Ered + 02 - k3 E,,P k4 2 - Eox + Pz

The rate equation arising from this scheme is

1 kz + k4 1 1 -=-------+ V k&4 kJglucose1 + k&&l

Since V,,, with infinite concentrations of oxygen and glucose is k&,/(lcz + k4) = 235 set-r, and kd has already been evaluated at 370 se?-‘, it follows that kz = 650 set-‘. The scheme thus accounts for all the experimental results currently available in terms of four constants.

Two points require comment. First, the identification of the first order steps characterized by kz and kq with the dissociation of product is admittedly arbitrary, and unsupported by any experimental evidence. An alternative, that lcz and kd represent intramolecular configurational changes, is also unsupported by experimental evidence, although several attempts have been made to secure it. In particular, there is no evidence of spectrophotometric changes in the regions of strong absorbance by the aromatic amino acids uncorrelated with changes in regions of strong absorbance by flavin. Further, as already mentioned, changes in fluorescence correlate closely with changes in flavin absorption, again suggesting an absence of changes involving the aromatic amino acids. Dyestuffs such as l-anilinonaph- thalene-8-sulfonic acid are nonfluorescent in aqueous solution, but fluoresce on adsorption (20). There was no evidence of a difference in adsorption of this dyestuff as between oxidized and reduced enzyme.

Second, the reactions have all been written as irreversible. The justification for this in the case of kl and lcl has already been given in discussing the half-reactions. The case of kz requires comment because of the evidence of Nakamura and Ogura (14) that glucono-6-lactone is inhibitory. The most obvious suggestion is that the lactone could reverse the second step of the reaction with glucose. This has been tested by the stopped flow method as well as in static spectrophotometric experiments. We have confirmed that the b-lactone is a weak inhibitor of glucose oxidation, but, whereas inhibition by reversal through a constant, i&, would result in the area under a curve such as that in Fig. 2 remaining constant (although the reaction would take longer), in practice the area was greater. It seems that the lactone is able either to stabilize the E,,H202 complex or to combine competitively but weakly with free oxidized enzyme. There is, therefore, no evidence for reversal by glucono-d-lactone.

It should be pointed out that the method described here for determining the values of kz and kq is far from being the only one. Several algebraic procedures have been developed which offer a test of specific schemes; they depend on the ratio of


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November 1964 Q. H. Gibson, B. E. P. Xwoboda, and V. Massey 3933

oxidized to reduced enzyme during turnover, and they may also be used to yield numerical values. Details of these procedures will be published separat,ely.

The scheme for glucose oxidation has been used to evaluate the results of experiments carried out at different temperatures, and the values of the four constants are given in Table I for four different temperatures. Although the data are scarcely sufficiently precise to allow values for the apparent activation energies of the four reactions to be given, these are strikingly different. In particular, the rate of dissociation of lactone increases so rapidly with temperature that above about 20” it no longer has sufficient influence on the rate to be determined. The rate of reaction of oxygen with the reduced enzyme, on the other hand, is almost independent of temperature.

The effect of temperature on the different steps in the reaction supplies a reason why Nakamura and Ogura (14) decided that the oxygen half-reaction was of the form Ered + O2 -4 E,, +

H202. All their experiments were carried out at room temperature, where the product dissociation stage is kinetically unim- portant. As mentioned in the introductory section, Nakamura and Ogura have described their results by the scheme

E,, + glucose G E,,glucose + Ered + P

E red + 02 + Em + &Oz

This scheme gives an accurate description of their experiments, and would fit all our own results at 20” or above, with the im- port,ant exception of the half-reaction experiments. Nakamura and Ogura did not do any half-reaction experiments with glucose, and so did not have an opportunity to observe that there is no limiting rate for this sugar.

One test of the schemes proposed for glucose and 2-deoxyglucose and of the values assigned to the various constants is to compare the calculated and observed inhibition of glucose oxidation by 2-deoxyglucose. The rate expression appropriate to this situation is

- ’ 0% + k,) +

k&D +

klG

klG + (ks + k,) k2 kskD kg + kr

+ K& 1 The constants are assigned according to the scheme

where G and D are glucose and 2-deoxyglucose, respectively. The agreement between theory and experiment (Fig. 5) is re- garded as a satisfactory confirmation of the accuracy of the kinetic experiments.

A further test of the over-all scheme with glucose is to use the rate constants to reproduce the spectrophotometric findings of a turnover experiment. This has been done in Fig. 7, where the lines were taken from the plotting table of an analogue computer (Solartron Electronic, Farnborough, Hants, England) and the points are experimental. The agreement is within experimental error and confirms the adequacy of the scheme proposed. It is an interesting consequence of the scheme that the first turnover

Spectrophotometric Turnover

0 f 0 I 2

Time (sec.)

FIG. 7. Comparison of the observed and calculated courses of the absorbance at 450 rnp during the reaction of glucose with oxygen (initial concentration, 6.8 X lo+ M) catalyzed by glucose oxidase (1.17 X 10e6 M) in the presence of 1 X 10e3 M KCN. The concentrations of glucose are shown by the lines, which were calculated by using a Solartron analogue computer. The points are experimental. The scheme was that given in the text with kl = 2.1 X lo3 M-I see-l, kz = 650 set-I, ka = 1.3 X lo6 M-I see-*, and kq = 370 set-1. Conditions as for Fig. 1.

of the enzyme after mixing must differ from all others, since all of the oxidized enzyme is initially free to react with glucose, while after the first turnover a proportion of the enzyme is bound to product (HzOz). The result is an overshoot of the steady state concentration of reduced enzyme. Some of the stopped flow reaction records show a spike which might possibly be interpreted in this way, but the high turnover rate, even at O”, makes this uncertain. Unequivocal evidence of a similar phenomenon has recently been obtained with the n-amino acid oxidiase (II), in which the low turnover rate makes the initial oscillation in absorbance easy to follow.

The very slow rates of oxidation of mannose, galactose, and xylose can be attributed directly to a glucose oxidase-catalyzed reaction. In the first place, Adams, Mast, and Free (21) have isolated the final products in the enzymic oxidation of galactose and mannose and shown these to be the corresponding aldonic acids. Thus galactose and mannose are oxidized directly to the aldonic acids without a prior conversion to glucose. Second, we have observed in anaerobic titration experiments that glucose oxidase flavin is very slowly but completely reduced by a 1 or 2 mole excess of galactose. This observation eliminates the possibility that the oxidation of galactose is due to a small contamination of galactose oxidase in our glucose oxidase. Similar arguments can be put forward for the other two sugars.

The results with the different sugars have been present.ed in terms of the simplest possible kinetic scheme adequate in each case. This does not imply that we consider the reaction mechanism necessarily to be different for each of the sugars.

A minimal general scheme accommodating all the results with all sugars would be

h Eox + S I

kz ka

k-1 EoxS - EredPl - Ered + PI

E red + 02 --++ EoxPz ks

- Eox + pz


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There is clear experimental evidence for the formation of an enzyme-substrate complex with 2-deoxyglucose. There is, however, no evidence for the step characterized by i&, presumably because kz << kl with this sugar, and in fact kz defines the rate- limiting step in the reaction.

With no other sugar is there evidence for an enzyme-substrate complex, and if such a complex does indeed exist, the Ks value must be of the order of at least 5 M.

It will be interesting, when the number of enzymes examined in detail by rapid kinetic methods is greater, to know how general is the formation of a detectable enzyme substrate complex. Certainly, as has already been recognized in principle by others, and documented experimentally here, Michaelis-Menten kinetic behavior in turnover experiments offers no guarantee of the formation of such a complex.

Although it is, of course, a truism that kinetic experiments can yield only kinetic and not chemical information, it is perhaps desirable to emphasize that the interpretation of the results presented here does not depend on the chemical nature of the enzyme intermediates involved. Thus, the absence of optical density changes at wave lengths longer than 500 rnp appears, at first sight, to rule‘ out the participation of a kinetically significant semiquinoid flavin form in catalysis. This conclusion, however, is based on the assumption that in enzymes, as in free flavins, the flavin semiquinone must have a long wave length absorption band. Results already presented (11) with n-amino acid oxidase have shown that it is possible to modify the absorption spectrum of free enzyme semiquinone through combination with a substrate radical. With glucose oxidase there is evidence that a similar combination may exist.4 We would reiterate, therefore, that the chemical nature of the form of the enzyme designated Ered

is still uncertain.

SUMMARY

1. The rates of reaction of glucose oxidase from Aspergillus

niger with the sugars glucose, 2-deoxyglucose, mannose, galactose, and xylose have been measured by manometric and stopped flow methods.

2. Glucose reacts much faster than the other sugars; V,,, at 0” = 240 see-l; K, glucose = 0.11 M, K, 0s = 2 X 10m4 M.

3. 2-Deoxyglucose gives V,,, at 0” = 15 set-l, K, 2-deoxyglucose = 0.025 M.

4. The rates with mannose, galactose, and xylose are directly proportional to sugar concentration at least up to 1 M sugar.

5. The rates of reduction of oxidized enzyme by sugars under anaerobic conditions (half-reactions) are: glucose, 1 X lo4 M-I set-l ; mannose, 22 i@ set?; galactose, 8 M-I see-l; and xylose, 3 ~-lsec+. The rates are accurately proportional to sugar concentration up to the highest concentrations which could be used, and there is no evidence of the formation of an enzyme- substrate complex.

4 B. E. P. Swoboda and V. Massey, unpublished observations.

6. The reaction of oxidized enzyme with 2-deoxyglucose is an exception. The half-reaction gives a limiting rate of 50 see-’ at 25’, 15 set-l at O”, and K, = 0.04 M at 25”, 0.025 M at 0”.

7. The rate of reaction of reduced enzyme with oxygen is 1.4 x 106 M-' set-l.

8. A scheme for analyzing spectrophotometric rapid reaction results is given. The oxidation of glucose is described by the scheme

E red + 02 * Ed’2 k4

- Eox + Pz

where P1 and Ps are gluconolactone and HzOz, respectively, and G is glucose. Steps 2 and 4 are first order, and contribute to the observed limiting rate at infinite substrate concentration. Their identification with dissociation of product is speculative.

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Quentin H. Gibson, Bennett E. P. Swoboda and Vincent MasseyKinetics and Mechanism of Action of Glucose Oxidase

1964, 239:3927-3934.J. Biol. Chem.

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Kinetics and Mechanism of Action of Glucose Oxidase* · Turnover of Enzyme-The results of manometric and stopped flow experiments are in satisfactory agreement, as illustrated in