KINETICS OF MALT a-AMYLASE ACTION*

BY SIGMUND SCHWIMMER

(From the Enzyme Research Division, Bureau of Agricukwal and Industrial Chemistry, Agricultural Research Administration, United States

Department of Agriculture, Albany, California)

(Received for publication, April 12, 1950)

The action of cw-amylase on undegraded starch and on dextrins of high molecular weight is roughly 100 times as rapid as its action on the smaller dextrins formed subsequently. The mechanism of this action has been studied in two ways. By kinetic methods, the course and rate of hydrolysis at varying substrate concentrations are determined. Thus Myrback and Johansson (1) calculated the affinities between the varying split-products and the enzyme from a study of the course of hydrolysis at different sub- strate concentrations. Similarly Bernfeld and Studer-P&ha (2) have in- vestigated the rate of action of various cr-amylases on amylopectin at varying substrate concentrations. Alternatively, the reaction can be stopped at different stages and the products characterized as to chain length, structure, and subsequent capacity for hydrolysis. Myrblick and Lunden (3) have made a substantial contribution with this approach. More recent studies include that of Alfin and Caldwell (4) on the digestion products of pancreatic amylase action on potato starch and on corn amylose. Swanson has recently characterized the products of action of salivary amylase on glycogen and on amylose (5). Perhaps the current view of the whole case has been succinctly expressed by Myrbllck (6) that cr-amylase “can attack substantially all normal glucosidic linkages in a chain molecule with velocities varying with the distance of the linkage in question from the end-groups.”

Three phases of the action of crystallized malt cr-amylase (7) are con- sidered in this report: (a) the ultimate extent of the hydrolysis of starch, (b) the change in the velocity of hydrolysis with increasing substrate con- centrations for a variety of substrates other than starch, and (c) inhibition by products of the reaction. It has been found that cu-amylase will ulti- mately hydrolyze 50 per cent of the glucosidic bonds, with maltose, glucose, and apparently trisaccharides as end-products. Calculation according to classical enzyme kinetics indicates that, for all substrates with chains more than 10 glucose units long, the affinity constant K is invariant within experiment.al limits. The amyloses are hydrolyzed at about double the maximum rate of the amylopectins. Results of the inhibition studies

* Enzyme Research Division Contribution No. 126. 181

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182 KINETICS OF a-AMYLASE ACTION

indicate that the end-products of cy-amylase action inhibit non-competi- tively, whereas incompletely hydrolyzed split-products compete with un- hydrolyzed substrate for the enzyme.

Materials and Methods

Initial Rate Studies-The substrates used in studying the initial rate are listed in Table I. The following samples of amylose were the same as those used by Potter and Hassid’ (8): potato, tapioca, Easter lily, corn, and acid-modified corn amyloses. The amylopectin samples and wheat amylose were prepared by the thymol method of Haworth, Peat, and Sagrott (10). The glycogen sample was obtained from a commercial source. The &limit dextrin was obtained by allowing crystalline P-amylase from sweet potatoes (11) to act on corn amylopectin until no further in- crease in reducing action was detected. The dextrins were precipitated from the digestion mixture by the addition of alcohol to 50 volumes per cent and then washed with alcohol and ether and dried.

The degree of polymerization was obtained by determining the reducing values of these substrates on dinitrosalicylic acid, as described below. For comparison, the degree of polymerization of the amyloses used is shown, as determined by Potter and Hassid (8, 9) from osmotic pressure measure- ments on the corresponding acetates. The average chain length determined by Potter and Hassid by means of periodic acid oxidation and the viscosities of these samples are also included in Table I.

It should be pointed out that the molecular weights of these substrates are still a matter of some disagreement. Thus Kerr and Cleveland (12) and Meyer et al. (13) have presented evidence that the molecular weights of the amylopectins are of the same order of magnitude as those of the amyloses.

For the determination of the initial velocities of hydrolysis, a 500 mg. sample of the substrate was dissolved in 40 ml. of 0.5 N NaOH. The pH was adjusted to 4.75 with 2 ml. of glacial acetic acid, and the volume of solution adjusted to 50 ml. Three different substrate concentrations were used. They contained 5.0,2.5, and 1.25 ml. of the stock substrate solution respectively, 0.0, 2.5, and 3.75 ml. of bucer (prepared as for the stock sub- strate), 4.9 ml. of saturated calcium sulfate solution, and 0.1 ml. of a 1: 100 dilution of stock enzyme solution to give a total of 10 ml. of digestion mixture. The stock enzyme was a solution of thrice crystallized malt a-amylase (7), containing 3.7 mg. of protein per ml. The final concentra- tion of cu-amylase per ml. of digestion mixture was calculated from the reported molecular weight of 59,500 (7) to be 6.2 X 1O-s PM of enzyme

1 The generous gift of these amylose samples. from Professor W; Z. Hassid of the University of California is gratefully acknowledged.

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8. SCHWIMMER 183

per ml. The substrate concentrations were calculated as 30.8, 15.4, and 7.7 PM of glucose existing in glucosidic linkages per ml. of digestion mixture?

Substrate hydrolysis at 30” was followed by determining at intervals the increase in the reducing action of 2 ml. of the digestion mixture on 1 ml. of dinitrosalicylic acid reagent3 (14). After boiling in a water bath for exactly 5 minutes, the reagent-sugar mixture was cooled, diluted to 10 ml. with distilled water, and its per cent transmission with a 540 ml.r filter was read in an Evelyn photoelectric calorimeter. A more complete series

TABLE I Substrates for a-Amylase Action

Substrates (amyloses)

1. Potato ........................ 2. Easter lily .................... 3. Tapioca ....................... 4. Corn .......................... 5. “ (acid-treated). ............ 6. Wheat ......................... 7. Potato amylopectin ........... 8. Corn ” ............. 9. Wheat “ .............

10. &Limit dextrin ............... 11. Potato starch, soluble ......... 12. a-Dextrin ..................... 13. Glycogen ......................

Degree of polymerization from

“;-&c;;g

1000 600

1250 760 400

200 10-11

Osmotic pressure*

930 620

1,300 800 400

(40,000) (30,000-37,000)

(25,000)

(40,000)

-

. .

-

930 620 650 270 130

15-25

Intrinsic viscosities’

1.95 1.06 2.25 1.23

* Samples 1 to 5 and data are those of Potter and Hassid (8, 9).

of concentration was run on one substrate, wheat amylose. Retrogradation was minimized by neutralizing each aliquot of the cold alkaline amylose solution within a few seconds before adding the enzyme.

Inhibition Studies-The substances used as inhibitors were (a) maltose hydrate, (b) the non-fermentable “limit dextrin” resulting from the com- bined action of cr- and fi-amylase on soluble starch (15), and (c) cr-dextrin, obtained by precipitation with alcohol from a digest of soluble starch by

*This method of expressing substrate concentration has been found very con- venient in the presentation of these results and is adhered to throughout the paper. It is regretted that no short term is available to express the idea behind the misnomer “moles of glucosidic bonds.”

*This sugar reagent contained 1.0 gm. of dinitrosalicylic acid and 30 gm. of Rochelle salt in 100 ml. of 0.6 N NaOH. The reaction was found to follow Beer’s law according to the relation M = 2.8L, where M = the microequivalents of maltose and L = the optical density of the solution of the reduced reagent.

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184 KINETICS OF a-AMYLASE ACTION

cw-amylase at the point where about 10 to 12 per cent splitting of total glucosidic bonds had occurred.

As the inhibitors are also reducing substances, total reducing values would be prohibitively large in comparison with the increase owing to hydrolysis. Accordingly, the rate of dextrinization was determined by the color of the digest with iodine. The method is essentially that described by Schwimmer (16), except that varying concentrations of substrate were used. In the present case 5 ml. of enzyme solution (2.1 units or 28 y of crystalline Lu-amylase) were added to 10 ml. of Lintner soluble starch at pH 4.75, containing also the amount of inhibitor indicated in Table IV. After incubation at 30” for various lengths of time, an aliquot of the digest was added to an iodine-potassium iodide mixture, the volume adjusted to 15.5 ml., and the color of the resulting dextrin-iodine complex read in an Evelyn photoelectric calorimeter at 660 mp. The aliquot was so taken, that the amount of total carbohydrate added to the iodine-iodide solution was kept constant, i.e., 5.76 mg. per ml. of the original solution of Lintner soluble starch. Thus the highest starch concentration used was 11.52 mg. per ml. of digest, and 0.50 ml. of the digest was added to the iodine solution; when half as much starch was used, 1 ml. of digest was added to the iodine solution, and so forth. 1 unit of enzyme is defined as that amount in the digestion mixture which in 10 minutes decreases the reading in the calorimeter to 50 per cent transmission at 660 rnp.

The relative rates of hydrolysis were then calculated from the apparent units obtained in this manner by dividing the latter by the appropriate dilution factor, since the units indicate the percentage change per unit of time rather than the actual amount of substrate changed. The rate at the highest starch concentration without inhibitor has been arbitrarily taken as 100. For example, when 11.52 mg. of substrate per ml. were used, the time required to obtain the standard color with 0.5 ml. of digestion mixture was 4.76 minutes, corresponding to 2.1 (10/4.76) units of enzyme; this velocity is thus equal to 100. When 5.76 mg. of substrate were used, 1 ml. of digest was added to the iodine-iodide solution and the time required to reach the same color value (after appropriate dilution) was 2.92 minutes. ,The apparent activity was therefore 3.42 units, the velocity was equal to one-half this value (1.71), and the relative velocity was 81.5.

Extent of Digestion-The extent of digestion was carried out in essentially the same manner as described before (7), the hypoiodite titration being used to determine the appearance of reducing sugars (17).

Graphic Analysis of Results-The classical dissociation constant of Michaelis and Menten (18) (K,) for enzyme-substrate complex formation and the maximum velocity (V) obtainable for a given enzyme concentra- tion are taken from the Lineweaver-Burk equation (19).

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S. SCHWIMMER 185

1 K, 1 -=- 0 v s +i 0

or VS

‘=K,+S

(1)

(1,a)

where v = the initial reaction rate and S = the substrate concentration. This equation is based upon the following assumptions: (1) the rate is proportional to the concentration of enzyme-substrate complex, and (2) the maximum rate is proportional to the total enzyme (e) present,

e = kV (2)

where Ic, when expressed in molecular terms, is a measure of the turnover number, and (3) the concentration, S, is essentially unchanged by com- bination with the enzyme.

When an inhibitor in concentration I competes with substrate for the enzyme (20), then

vo K, I --=I+ x, K,+S VI

(3)

where vo is the velocity in the absence, vl is the velocity in the presence of I at concentration X, and Kr is the dissociation constant for enzyme-in- hibitor complex. When the inhibition is non-competitive,

vo -=1+& VI

(4)

lrZesult.3

The most complete set of data was obtained with wheat amylose. Its hydrolysis at varying substrate concentrations as a function of time is shown in Fig. 1, and the initial rates of hydrolysis are listed in Table II. The variables l/v and l/S are plotted against each other in Fig. 2. The straight line was obtained by applying the method of least squares to the data. The absolute value of the x: intercept is the reciprocal of the K, value (4.8 microequivalents of glucosidic bonds per ml.) ; the value of the y intercept is the reciprocal of the V value for wheat amylose within ex- perimental error (0.26 microequivalent of glucosidic bonds hydrolyzed per ml. per minute).

Data for the other substrates were subjected to the same treatment, with the limitation imposed upon them that the K, values were all assumed to be equal to 4.8. Table III contains initial velocity for these substrates along with the V values obtained graphically (except for B-limit dextrin and glycogen). The curves for theoretical initial velocity versus substrate

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186 KINETICS OF a-AMYLASE ACTION

concentration obtained for each substrate with these constants and Equa- tion 1, a are shown in Fig. 3. With K, = 4.8, these were the best fits

30.8

TIME IN MINUTES FIQ. 1. The initial course of hydrolysis of wheat amylose by malt a-amylase at

varying substrate concentrations; the initial concentration of substrate (expressed as microequivalent of glucose in glucosidic linkage per ml. of digestion mixture) is shown for each curve.

TABLE II Initial Velocity of Hydrolysis of Wheat Amylose by Malt a-Amylase

S’ n* S 0 s 0

30.8 0.232 11.55 0.187 3.85 0.115 23.1 0.214 7.70 0.158 3.08 0.099 15.4 0.206 5.39 0.135 2.31 0.086

* S and v are expressed in terms of microequivalents of bonds per ml. and micro- equivalents of bonds hydrolyzed per ml. per minute, respectively.

obtainable for the experimental data, except for the values obtained with the substrates P-limit dextrin and glycogen. With j3-limit dextrin the

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S. SCHWIMMER 187

best curve was obtained when K, = 6.9 PM of glucosidic bonds per ml. and V = 0.20 PM of bonds hydrolyzed per ml. per minute. For glycogen the corresponding values were K, = 20 and V = 0.14.

The course and extent of hydrolysis of soluble starch at varying con- centrations of ar-amylase and in the presence of crystalline sweet potato /3-amylase are shown in Fig. 4. As can be seen, the ultimate degree of hydrolysis never seems to have exceeded 50 per cent hydrolysis of the glucosidic bonds. However, qualitative tests (modified Barfoed test and osazone formation) indicated the presence of glucose in the hydrolysates. Hence probably some trisaccharide was also present to balance the excess over 50 per cent hydrolysis.

I/sx100 FIG. 2. Plot of l/v against l/S for wheat amylose

The relative rates of hydrolysis of soluble starch as functions of substrate and inhibitor concentration are shown in Table IV. A plot of the pertinent variables (l/v0 against l/S) is presented in Fig. 5,~. The relation between the reciprocal of the fractional inhibition (v~/Q) and the inhibitor concen- tration for the a-dextrin (Fig. 5, b), maltose (Fig. 5, c), and limit dextrin (Fig. 5, d) reveals some rather interesting differences. Thus maltose seems to inhibit non-competitively over the whole range of substrate concentra- tion, whereas the limit and ar-dextrins exhibit considerable inhibitory action only at the lower substrate concentrations. At these lower con- centrations, the limit dextrin seems to inhibit non-competitively, the ar-dextrin competitively. Demonstration of this competition is shown by the constancy of the.value for the inhibition constant Kr, when Equation 3 is applied. From the calculated value of K, = 2.77 mg. per ml. from

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188 KINETICS OF a-AMYLASE ACTION

Fig. 5, a, t,he corresponding values for KI at substrate concentrations equal to 1.44 and 2.88 mg. per ml. are 6.1 and 6.4 mg. per ml. of inhibitor respectively. At a substrate concentration of 5.76 mg. per ml. the cor- responding value for K, is equal to 12.5 mg. per ml., thus showing both the decreased inhibitory action and the indeterminate nature of the competition at a higher substrate concentration. Application of the equation for non- competitive inhibition (Equation 4) to the limit dextrin and to maltose

0 IO 20 30

S FIQ. 3. Velocity (v) of hydrolysis of various substrates as a function of substrate

concentration. The numbers in the graph identify the substrates as listed in Table I. The points (0) represent the experimentally determined values of v for all sub- strates except p-limit dextrin (X) and glycogen (0). The curves represent the cal- culated v values from Equation 1, a, with an assumed K, value of 4.8 and V values as described in the text. 0 shows the calculated curve for glycogen with the values shown in Table III.

yields inhibition constants equal to 27 and 57 mg. per ml., respectively. Thus the inhibitory activity of these end-products increases in the order of maltose, limit dextrin, and a-dextrin.

DISCUSSION

The conclusion that the K, values are constant for all but two of the substrates tested is based, of course, on the assumption that a measurable K, exists for each substrate. Except for wheat amylose, the velocity of hydrolysis of these substrates was determined when the enzyme was

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$3. SCHWJMMER

TABLE III Constants for a-Amylase Action

,---_-r_. ..-l_-l___ Initial velocities (e)

Substrate - 1 !

-- “I

Potato ....................... Easter lily ................... Tapioca ....................... Corn ........................

‘C amylose acid-treated. Wheat amylose. ............. Potato amylopectin. ......... Corn “ .......... Wheat “ .......... O-Limit dextrin. ............. Lintner soluble starch ........ a-Dextrin. ................... Glycogen ..........................

S’ = 30.1

0.36 0.33 0.27 0.27 0.26 0.23 0.17 0.16 0.16 0.16 0.16 0.14 0.09

T i = 15. F = 7.1

0.31 0.26 4.8 0.29 0.24 4.8 0.26 0.20 4.8 0.23 0.18 4.8 0.21 0.17 4.8 0.21 0.16 4.8 0.15 0.12 4.8 0.14 0.11 4.8 0.14 0.11 4.8 0.13 0.10 6.9 0.14 0.11 4.8 0.13 0.10 4.8 0.06 o.o* 20.0

--

V L x 10-4

A ---

0.42 6.8 0.39 6.3, 0.32 5.2 0.39 4.9

.0.23 4.6 0.26 4.2 0.29 3.3 0.19 3.1 0.19 3.1 0.20 3.3 0.18 2.9 0.16 2.6 0.14 2.3

189

* S and K, are expressed as microequivalents of bonds per ml., v and V as micro- equivalents of bonds hydrolyzed per ml. per minute, and k as microequivalents of bonds hydrolyzed per micromole of a-amylase per minute.

0 I 2 3 4 5 6. 7 8

TIME-HOURS FIN. 4. Course and extent of hydrolysis of 2 per cent soluble starch by 1, 15, and

170 a-amylase units at 33”. 0 represents hydrolysis obtained with 170 units of 01- amylase plus 10 units of sweet potato &amylase.

evidently approaching saturation with substrate (see Fig. 3). However, this assumption is not unreasonable, since it has been rigorously proved for wheat amylose, for which substrate concentrations in the region of the K, values were used. Furthermore, the calculated values based on this

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190 KINETICS OF a-AMYLASE ACTION

assumption fitted the experimental values fairly well. It should be pointed out that constancy of K, on a gm. or equivalence basis actually means that K, on a molecular basis (which presumably would have more signifi- cance with regard to mechanism) is related to K, on a gm. basis as follows: K. (mblecular) = K, (gm.)/(mol. wt.). Thus the afhnity of the enzyme for substrate is directly proportional to the molecular weight. Obviously

1.5c > \

>O

LOC

26

5 2c

co ,c \ ..

IC

)-

IL

,-

)-

,-

I- /

J (a ,

IO 20 30

I

/

10

(a) I I I

a2 0.4 0.6

I/S

3 6

I FIG. 5. Summary of the effect of inhibitor concentration (I) on the rate of dextrini-

z&ion of soluble starch by malt cu-amylase at varying substrate concentration (the symbols are defined in the text). The substrate concentrations are as follows: 11.52 (O), 5.76 (O), 2.88 (A), and 1.44 (Cl), mg. per ml. respectively.

the data have significance with respect to the above relation only if the substrates used do have different molecular weights.

The K. value found for almost all the substrates is of the same order of magnitude as that found by Bernfeld and Studer-P&ha (2), using pan- creatic and bacterial amylase acting on amylopectin, and that of Myrback and Johansson (l), using malt cr-amylass acting on soluble starch. The apparent independence of the size and structure of the substrate exhibited by the K, values indicates that the essential substrate for cr-amylase is the “glucosidic” linkage rather than the whole molecule; i.e., one linkage is as susceptible as another to combination with enzyme, provided the

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S. SCHWIMMER 191

linkage is in a straight chain of at least 10 glucose units. Thus glycogen according to Meyer and Fuld (21) does not contain any chains greater than 7 units long. Whereas the “exterior” chains of amylopectin are 15 units long, the “interior” chains, the only ones present in P-limit dextrin, aver- age about 7 to 8 glucose residues (13). This may explain why these two substrates have K. values higher than the other substrates tested. The great decrease in velocity of hydrolysis of starch which occurs when the fragments average about 8 units and lower is also in accord with the con- cept that K, values are independent of chain length and structure when above about 10 units.

On the other hand the V values seem to depend in some way on both the molecular weight and degree of branching. Thus the amyloses hydro-

TABLE IV E$ect of Digestion Products on Velocity of Deztrinization of Soluble Starch by

Inhibitor

-

E

None Maltose

Limit dextrin

a-Dextrin

.-

-

Malt CY-AZ 4 -

Inhibitor :oncentration, mg. per ml

‘lase

Substrate concentration, mg. per ml.

11.52 1 5.76 1 2.88 1 1.44

Relative hydrolysis velocities

0.00 100 6.35 90

12.70 82 25.40 67

6.68 102 13.35 106 26.70 100

1.67 105 3.34 100 6.68 100

-

-

66 57 80 81 84 78 76 70

-

-

56 39 50 36 46 31 38 27 44 31 38 27 29 19 49 32 45 28 37 23

lyze at about twice the rate of the amylopectins. If the differences in V within the amylose series are to be considered significant, then we see that the highest velocity is obtained with the least branched and most highly polymerized substrate (Table I). The reason for these differences is not immediately apparent.

It is of interest to note that the hydrolysis of wheat amylose follomied a zero order reaction curve at very low substrate concentrations (Fig. 1). Simple enzyme-substrate combination predicts that the reaction should be first order at these concentrations, since the enzyme is not saturated with the initial substrate at these low concentrations. However, if one assumes that the enzyme Pecombines successively with smaller and smaller split- products, and with equal affinities for these split-products, then the enzyme

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192 KINETICS OF a-AMYL.ASB ACTION

will be combined to at least as great a degree after time t as at zero time. The reaction will then follow zero order kinetics. This supposition is in complete agreement with the finding that t,he K, values are constant for all substrates whose chain length is greater than about 10 glucose units. It also explains why the course of hydrolysis is always zero order to the point of appearance of large amounts of the smaller dextrins.

One would expect a priori that substances which can combine with a-amylase to form a hydrolyzable enzyme-substrate complex would com- pete with another substrate (viz. soluble starch) for active sites on the enzyme. This in fact was found for the inhibition with a-dextrin, which can be further hydrolyzed by the enzyme. On the other hand, unhydro- lyzable or difficultly hydrolyzable end-products (for example maltose) were found to inhibit in a non-competitive manner.

There may be significance in the finding that the ar-dextrin, whose K, value falls within the same range as that of the majority of the substrates, inhibits competitively, whereas substances less than 10 glucose units long (maltose, “limit” dextrin) inhibit non-competitively.

Previous studies on the extent of hydrolysis of starch with wamylase usually show that the “second” slow phase of hydrolysis has no defi- nite end-point, but continues with lessening velocity until about 45 to 50 per cent of the linkages are split (22). We have definitely shown that by using a high enough concentration of enzyme the hydrolysis can pro- ceed within a comparatively short time to 50 per cent, when it apparently stops. The implied presence of trisaccharides in the final hydrolysate may explain why no “isomaltose” is found in hydrolysates from malt amylase action (23). From this point of view at least, malt a-amylase differs from fungal amylase action (24).

SUMMARY

The initial rates of hydrolysis by crystalline malt ar-amylase have been determined at varying concentrations of some amyloses, amylopectins, dextrins, and of glycogen. The inhibition of starch hydrolysis by products of the reaction has also been investigated. The amyloses were hydrolyzed at maximum rates which are about twice the maximum rates found for the amylopectins and glycogen. The highest maximum velocity (correspond- ing to a turnover number of 68,000) was obtained with amyloses which did not contain branch points.

Calculations, according to the classical Michaelis-Menten theory, re- vealed that, within experimental error, the affinities of almost all of the substrates for the enzyme showed a constant value on a “gm.” or “equiva- lent” basis. On a molecular basis, the affinity of the enzyme for the sub- strate is directly proportional to the molecular weight. Only the “affinity”

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S. SCHWIMMER 193

constants for glycogen and probably P-limit dextrin were significantly lower than for the other substrates. It is suggested that lowered af- finities are apparent only in substrates whose chain length is less than approximately 10 glucose units. This conclusion also agrees with the results of inhibition studies which indicate that the end-products of CY- amylase action inhibit non-competitively, whereas incompletely hydrolyzed split-products compete with starch for the enzyme.

The ultimate extent of the hydrolysis of starch by malt a-amylase was found to be at 50 per cent splitting of the total glucosidic linkages. Mal- tose, glucose, and trisaccharides are the apparent end-products.

BIBLIOGRAPHY

1. Myrbilck, K., and Johansson, N. O., Ark. Kemi, Mineral. o. Geol., 20 A, No. 6 (1945).

2. Bernfeld, P., and Studer-P&ha, H., Heb. chim. acta, 30, 1994 (1947). 3. Myrblick, K., and Lund&r, R., Ark. Kemi, Mineral. o. Geol., 23 A, No. 7 (1946). 4. Alfin, R. B., and Caldwell, M. L., J. Am. Chem. Sot., 71, 128 (1949). 5. Swanson, M. A., J. Biol. Chem., 172,805 (1948). 6. Myrbiick, K., Arch. Biochem., 14, 53 (1947). 7. Schwimmer, S., and Balls, A. K., J. Biol. Chem., 179, 1063 (1949). 8. Potter, A. L., and Hassid, W. Z., J. Am. Chem. Sot., 70,3774 (1948). 9. Potter, A. L., and Hassid, W. Z., J. Am. Chem. Sot., 70,348S (1948).

10. Haworth, W. N., Peat, S., and Sagrott, P. E., Nature, 167, 19 (1946). 11. Balls, A. K., Walden, M. K., and Thompson, R. R., J. Biol. Chem., 173,9 (1948). 12. Kerr, R. W., and Cleveland, F. C., J. Am. Chem. SOL, 71,3455 (1949). 13. Meyer, K. H., Bernfeld, P., Rathgeb, P., and Gtirtler, P., Helv. chim. acta, 31,

1536 (1948). 14. Bernfeld, P., and Studer-P&ha, H., Helv. chim. acta, 30, 1895 (1947). 15. Kneen, E., and Spoerl, J. M., Proc. Am. Sot. Brewing Ch.em., 20 (1948). 16. Schwimmer, S., Cereal Chem., 24, 315 (1947). 17. Jansen, E. F., and MacDonnell, L. R., Arch. Biochem., 8,97 (1945). 18. Michaelis, L., and Menten, M. L., Biochem. Z., 49,333 (1913). 19. Lineweaver, H., and Burk, D., J. Am. Chem. Sot., 66,658 (1934). 20. Ebersole, E. R., Guttentag, C., and Wilson, P. W., Arch. Biochem., 3,399 (1943-

44). 21. Meyer, K. H., and Fuld, M., Helv. chim. acta, 24,375 (1941). 22. Myrback, K., and Sihlbom, L., Ark. Kemi, 1, 1 (1949). 23. Wolfrom, M. L., Georges, L. W., Thompson, A., and Miller, I. L., J. Am. Chem.

Sot., 71, 2873 (1949). 24. Montgomery, E. M., Weakley, F. B., and Hilbert, G. E., J. Am. Chem. Sot., 69,

2249 (1947).

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Sigmund SchwimmerACTION

-AMYLASEαKINETICS OF MALT

1950, 186:181-193.J. Biol. Chem. 

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