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Multiple attack hypothesis of α-amylase action: Action of porcine pancreatic, human salivary, and Aspergillus oryzae α-amylases
BRCHIVES
OF
Multiple
BIOCHEMISTRY
Attack
Pancreatic,
AND
Hypothesis
Human
122, 8-16 (1967)
RIOPHYSICS
of cr-Amylase
Salivary,
JOHN F. ROBYT Department
of Biochemistry Received
and
Aspergillus DEXTER
AND
and Biophysics, February
Action:
Iowa
Action
oryzae
oc-Amyloses’
FRENCH
State University,
23,1967; accepted
of Porcine
Ames, Iowa 50010
May 5, 1967
Three conceptual action patterns of ol-amylase hydrolysis of amylose have been considered: single chain, multichain, and multiple attack. To test these concepts, curves were obtained relating the drop in amylose-iodine color to the increase in reducing value for amylolysis by human salivary (HS), porcine pancreatic (PP), Aspergillus oryzae (AO) a-amylases, and 1 M HzSO~. The observed differences in the curves for the amylases could only be interpreted as due to differences in degree of multiple attack. To test these concepts further, amylase digests at various stages of hydrolysis were separated by ethanol precipitation into polysaccharide and oligosaccharide fractions. The degree of multiple attack was determined from the ratio of the reducing value of the oligosaccharide fraction to that of the polysaccharide fraction. Under optimal conditions of pH and temperature, PP had a degree of multiple attack of 6, three times that of HS or AO. At pH 4.5, the degree of multiple attack of PP did not change, although its activity was reduced lo-fold. At pH 10.5, however, the degree of multiple attack was reduced to 0.7, approaching a multichain pattern.
It is widely believed that a-amylases from a number of sources hydrolyze starch at random points in the polymer chain to give a random distribution of products that eventually become identical (l-3). In 1953, Kung et al. (4) published a series of curves relating the drop in iodine color (blue value2) to the increase in reducing value for the action of several a-amylases upon amylose. These curves varied significantly for the individual a-amylases, thus indicat,ing that the act’ions of a-amylases were not
identical. Kung et al. (4) suggested that the differences were due to the different 01amylases hydrolyzing amylose into different chain lengths. Since then, paper chromatographic analyses have shown that, in fact, a number of or-amylases produce low molecular weight products with molecular size distributions characteristic of the individual enzymes (5-S). Chromatographic analysis of HS- and PP-amylases digests show (Fig. 1) that these two amylases produce very similar products. Therefore, their widely different blue valuereducing value curves (Fig. 2) cannot be accommodated by the explanation advanced by Kung et al. (4).
1 Journal Paper No. J-5460 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Projects 1116 and 1485. Support’ed by grants from the Corn Industries Research Foundation, NIH GM-08822 and ARS 12-14-100-l-7668 (71). Presented in preliminary form at the 150th meeting of the American Chemical Society, IXvision of Biological Chemistry (Abstracts 96C, 1965). 2 Blue value is defined as (At/A0 X 100, where A, and AL are the absorbancies (620 rnp) of the iodine complex of the digest at zero time and at t minutes of hydrolysis.
For strates
enzymic action there are three
on polymeric distinct action
subpat-
terns: single chain, multichain, and multiple attack. In the single chain action, once the enzyme forms an active enzyme-sub3 Abbreviations used: PP, porcine pancreatic a-amylase; HS, human salivary my-amylase; AO, Aspergillus oryzae or-amylase (Taka A).
8
tiuman
sal~varv
~i-Amvlase pti
6 9. 40’
Ilmu/mli C,
dqest 1OmM
of
Amvlose
11 W/ml1
chloride
porcine
Pancreatlc
(1 mu/ml) C
1OmM
digest
of Amvlose
II
mg/mlb
chloride
0
FIG. 1. Time sequence chromatographic analysis of human salivary and porcine pancreatic a-amylase action on amylose. The conditions of the digests were: amylose (1 mg/ml); 20 mM, pH 6.9 sodium glycerophosphate buffer; 10 mM NaCl; 1 mU of enzyme/ml. Aliquots (100 ~1) were placed on the chromatogram. The first two columns represent cont.rols; thereafter a time sequence in minutes and hours. The chromatograms were obtained by the techniques of French el al. (34).
strate (E-S) complex, it catalyzes reaction in a “zipper” fashion toward one end of the chain and does not form an active complex with another substrate, until it comes to the end of the first chain.4 The multichain process is the classical random action in which the enzyme catalyzes the hydrolysis of only one bond per effective encounter. In the multiple-attack act’ion, once the enzyme forms an enzyme-polymer complex, in which there is suitable geometry for catalysis, the enzyme may catalyze the hydrolysis of several bonds before it dissociates and forms a new active complex with another polymer chain. The cleqreeof mult’iple attack may be defined as the average number of catalytic events, following the first, during t’he lifetime of an individual E-S complex. The single chain and multichain mechanisms 4 There is the possibility of more than one active site per enzyme molecule. In this case, the enzyme could form a complex with another substrate molecule at the second active site. However, if the two active sites were independent, it would be assumed that the individual active site would not form a complex with a second polymer until it had come to the end of the first chain.
represent the two extreme cases of polymer degradation; the multiple-attack mechanism is a more general concept including multichain and single chain mechanisms a,s extreme special cases. Up unt’il now, these concepts have been applied primarily to exoenzymes, e.g., pamylase and phosphorylase (9, 10). It has been shown that p-amylase has a multiple attack action (11, 12). a-Amylases, by contrast’, are generally regarded as being endoenzymes, t,hat is, enzymes that initiate attack at, random interior positions along the polymer chain. It is obvious that the gross facets of action pattern must be different for exo- and endo-enzymes. However, if one examines the details of action, it is clear t’hat the three concepts, originally developed for exo-enzymes, may a’lso be applied to the action of endo-enzymes (cf. Fig. 3). In the present investigation, we have devised an experimental method for measuring the degree of multiple attack, and have shown that various or-amylases exhibit varying degrees of multiple attack. Also, we have shown that t’he degree of multiple
10
ROBYT
60
AND
FRENCH
-
BLUE VALUE
30 -
40
-
30
-
20
-
P P (p H 4.5 I’\\
IO -
0
2
4
6
9
REDUdlNG
IO
I2
14
VALUE
I6
I6
AS
20
22
24
% APPARENT
26
28
30
32
34
MALTOSE
FIG. 2. Comparison of the drop in iodine color (blue value) with the increase in reducing value for the hydrolysis of amylose by various catalysts. Blue value is defined as (A JA,) X 100, where L4a and A, are the absorbancies (620 rnp) of the iodine complex of the digest at zero time and at t minutes of hydrolysis. (0) PP (pH 6.9), porcine pancreatic a-amylase at pH 6.9, 10 mM Cl-, and 40”; (0) HS, human salivary a-amylase at pH 6.9, 10 mM Cl-, and 40”; (n) PP (pH 4.5), porcine pancreat.ic ol-amylase at pH 4.5, 10 mM Cl-, and 40”; (A) AO, 4spergiZZu.s oryzae a-amylase at pH 5.5, and 40”; (A) 1 M sulfuric acid at 60”; (0) PP (pH 10.5) porcine pancreatic ol-amylase at pH 10.5, 10 mM Cl-, and 40”.
attack depends on t$he reaction conditions. For, example, with PP under optimal conditions (pH 6.9), the degree of multiple attack is about 6; at pH 10.5, the degree of
multiple attack drops to essentially zero, which corresponds to the multichain mechanism. With HS under opt,imal conditions, the degree of multiple att,ack is only about 3;
MULTIPLE
A
SINGLE
B
MULTIPLE
ATTACK
BY wAMYLASES
CHAIN
ATTACK
C. MULTI CHAIN
E,
FIG. 3. Types of attack action patterns for endo amylases. Three cases are presented; each case illustrates the action of a single enzyme molecule. The arrows represent the catalytic hydrolysis of a glycosidic bond; 0 represents an amylose molecule made up of glucosyl units linked 01, 1 + 4; + represents a reducing hemiacetal end group. The numbers refer t,o the sequence of hydroIytic events by the enzyme. The oligosaccharide product specificity in cases A and B is arbitrarily assumed to be maltose and maltotriose. The authors have assumed a definite polarity of the enzyme action toward the reducing end. However, the actual direction of the action is not known; it may as well be toward the nonreducing end or the enzyme may not have a preference, although the authors are inclined to believe that the action is in one direction or the other. For illustrative purposes the amylose molecule however, it undoubtedly possesses a certain amount of is pictured as a long “string”; secondary helical structure (35) that is not illustrated.
t,herefore, we int,erpret t)he blue value-reducing value curve differences as reflecting primarily differences in the degree of multiple attack by t*hese otherwise very similar a-amylases. EXPERIMENTAL
PROCEDURE
Analytical meihods. Reducing values were measured by the alkaline ferricyanide-cyanide procedure (13) with the Technicon Autoanalyzer. The method was not affected by the chain length of the maltodextrins (14) since equal reducing values were obt,ained by equimolar amounts of and maltotriose. This result glucose, maltose,
indicates that the method is a stoichiometric measure of the hemiacetal groups (reducing groups) present in a maltodextrin sample. In the present work, it has been assumed that themethod will similarly determine the hemiacetal groups for higher saccharides, e.g., 4-1000 glucose units. The blue value was determined by adapting the conditions of McCready and Hassid (15) for use wit,h the Technicon AutoanalyJer (16). Total carbohydrate was determined by t,he phenolsulfuric acid procedure adapted for use with the Technicon Autoanalyzer (16). Substrate. Superlose, a commercial amylose from Stein-Hall & Co., New York, was recrystallized twice with 1-butanol; 50 gm of Superlose
12
ROBYT
AND
was suspended in 500 ml of dimethyl sulfoxide and stirred until dissolved (about 10 hours). The dimethyl sulfoxide solution was diluted with 4.5 liters of water, and 200 ml of I-but,anol was added. The mixture was stirred overnight and centrifuged. The butanol-amylose complex was redissolved by adding small amounts to 5 liters of boiling water. After the complex was dissolved, and the solution had cooled to about 65”, 200 ml of 1-butanol was added, and the mixture was stirred overnight. The butanol-amylose complex was recovered by centrifugation and successively treated five times with 250 ml of anhydrous acetone. The average degree of polymerization (DP) of the recrystallized amylose, as determined by reducing value and phenol-sulfuric acid analysis, was 1000 f 50. Enzymes. Human salivary a-amylase (HS) was crystallized according to the method of Fischer and Stein (17). Porcine pancreatic cu-amylase (PP), crystallized twice by the method of Caldwell et al. (18), was obtained from Worthington Biochemicals, Freehold, New Jersey. Aspergillus oryzae a-amylase (AO) was crystallized twice according to the method of Fischer and DeMontmollin (19). Enzyme activity was measured by determination of reducing values using the ferricyanide method (13) or the Nelson copper method (20). Activity is expressed in International Units, viz., one unit (U) is that amount of enzyme that will hydrolyze one pmole of bond per minute at 40” and the optimal pH of the enzyme. Digests. Enzymatic digests. Amylose (124 mg) was dissolved in 2 ml of dimethyl sulfoxide (25”; about 30 minutes); this was diluted with 12 ml of 200 rnM buffer (pH 6.9 and 5.5, sodium glycerophosphate; pH 10.5, sodium glycinate; pH 4.5, acetate), 12 ml of 100 mM sodium chloride, and diluted to 120 ml with water. Enzyme was added to initiate the reaction: 300 mU at the optimal pH values (6.9 or 5.5), and 6.0 U at unfavorably low or high pH (4.5 and 10.5). The digests were incubated at 40”. Duplicate 5-ml aliquots were taken at various times over 120 minutes and added to 0.1 of 5 M trichloroacetic acid (for digests at pH 10.5,0.2 ml of acid was used). After standing approximately 30 minutes, the samples were neutralized with 5 M sodium hydroxide to pH 6 (pH paper). Acid digests. Amylose (210 mg) was dissolved in 2 ml of dimethyl sulfoxide and diluted to 100 ml with water; after equilibration at BO”, 50 ml of 3 M sulfuric acid was added; at various intervals 5-ml aliquots were taken in pairs, placed in an ice bath, and neutralized with 2 ml of 5 M sodium hydroxide to pH 6 (pH paper). Bernfeld (21) has suggested that retrogradation of amylose might be the cause for the differences observed for the blue value-reducing value curves
FRENCH of Kung et al. (4). With the concentration of amylose used in our digests (1 mg/ml), the formation of insoluble precipitates was never observed, and the carbohydrate analysis for each aliquot remained constant. In addition, identical curves were obtained by using different concentrations of amylase with a consequent sampling at different 1engt)hs of time. Since retrogradation is a function of substrate concentration and time, it would be impossible to get reproducible curves if retrogradation were taking place during amylolysis. Fractionation of enzyme digests by ethanol precipilation. Two volumes of anhydrous ethanol were added to one of the two 5-ml aliquots taken at various times. The samples were placed at 5” overnight. The resulting precipitates were centrifuged and washed twice with 5 ml of 70% ethanol and once with 5 ml of anydrous ethanol. The precipitate was then dissolved in 0.2 ml of dimethyl sulfoxide and diluted with 5 ml of water. The ethanol precipitates did not contain any small maltodextrins in the range of glucose to maltododecaose as judged by paper chromatographic analysis. Under the conditions of the experiment, the smallest polymer that could be precipitated had an average degree of polymerization of 20 glucose units. The reducing value and the carbohydrate COIIt,ent were determined for each ethanol-precipitated sample, from which the average DP could be calculated. The reducing value, blue value, and carbohydrate content were determined for each of the other aliquots that were not treated with ethanol. The degree of multiple attack (Table I) was calculated from the experimental data, as explained in the Discussion. DISCUSSION
The blue value versus reducing value curves (Fig. 2) show that for an equal drop in blue value (e.g., 60%) the three ar-a’mylases PP, HS, and A0 have different percentage conversions to apparent’ maltose when operating under optimum conditions. These differences in reducing values may be interpreted as differences in the degree of multiple attack as follows. Let us imagine that the intial substrate contains 1000 glucose units and that a certain enzyme encounters the substrate in the center of the chain and cleaves it into two fragments of 500 units each. If five additional glucosidic bonds are broken from one of the fragments to give five maltose molecules, the polymer fragment would be left with 490 units. If, with a different enzyme, ten bonds are
MULTIPLE
ATTACK
broken to give ten molecules of maltose, the polymer fragment would have 450 units remaining. The total blue value of t’he 500 + 480 fragments would be very nearly the same as that of the 500 + 490 fragments (23). However, t,he reducing value of t’he digest forming t,en maltose molecules would be almost t,wice that of the digest forming five maltose molecules. Thus, differences in reducing values for two enzyme digest,s having identical blue values point’ st’rongly to differences in the degree of multiple attack. The argument is lit)tle affected if maltotriose or maltotetraose result inst’ead of maltose. The data of Fig. 2, therefore, show qualitatively that the degree of multiple &tack decreases in the following order: PP (pH 6.9 and 4.5), HS, AO, and PI’ (pH 10.5). Acid hydrolysis, by its very nature, should give a degree of multiple attack of zero. Hence one might expect that it would show the lowest reducing value for an equivalent’ drop in blue value. In fact, however, the &ion of PI’ at pH 10.5 (Fig. 2) shows a lower reducing value than acid hydrolysis at equal blue values, with the differences becoming more marked as the reactions proceed. These differences must reflect (a) t#he avoidance of chain ends by PP, so that no glucose is produced, and (b) the preferent,ial attack by acid on the terminal linkages, thus producing somewhat, more glucose than in a purely random process (24). These “end-effects” are of litt#le significance during t,he early stages of the hydrolysis when the average size of the polymer molecules is large. The initial coincidence of t,lre pH 10.5 PI’ curve with that of acid hydrolysis is compatible wit,h the idea that t,he i&al a&& by a-amglase is at’ a random position along t’he polymer chain. On the other hand, after extensive hydrolysis, end-effects substantially modify the action pnt,tern and finally determine the various final product dist,rlbuOions found with va.rious cr-amylnses. The rationale behind the calculat’ion of t,he degree of multiple attack from the ethanol fractionatjion experiment is as follows. The tot’al increase in reducing power represents the sum of two types of bond cleavages:
BY wAMYLASES
13
(a) primary attack on a polyasccharide molecule to give two macromolecular (polysaccharide) fragments, and (b) secondary attack on t’he newly formed end of one polymer fragment t’o give low molecular weight oligosaccharides. The polymers may be easily separated from the oligosaccharides by ethanol precipitation. Kew reducing groups in the polysaccharide fraction are equal in number to the number of effective encounters. New reducing groups in the oligosaccharide fraction are equal in number to the number of bonds broken in secondary phases of multiple attack. The quotient T of the total reducing value, which is a measure of t,he total number of bonds broken, divided by the reducing value of the ethanol precipitate, which is a measure of the number of effective encounters, gives t’he number of bonds broken per effective encounter. Since the first bond broken releases a polymer fragment,, the average number of subsequen~t bonds broken (~-1) is numerically equal to the degree of multiple attack. The experimentally determined value, T, remained relatively constant until the blue value had dropped to around 50 74. Only samples whose blue value had not dropped below 50 C/owere used in t,he analyses, The increase in 1’ in the later stages of the reacGon is expected because of the formation of small polymers that arc: not, completely precipitated by C,S% ethanol. In this analysis it must be assumed t,hat formation of oligosaccharides through primary attack is insignificant. This is obviously a better assumption in the initial phases of hhe react,ion when the DP of the subst,rate is many hundreds or t,housands. In the later stages of the reaction, when t,he average DP of the polymeric substrate is less t,han 30-40, the assumption breaks down altogether. The results of the ethanol fract)ionation experiments (Table I) give a quantitative index for the number of attacks occurring per effective encounter. The data confirm t’he interpretation of the blue value-reducing value curves (Fig. 2). For PP, the degree of mult’iple attack (~-1) is A; that is, for every effective encount’er, PP on the average breaks six bonds and releases maltose, maltot,riose, or malt,ot,etraose. For HS, the
14
ROBYT
AND
degree of multiple attack is 2; for AO, 1.7; and for PP (pH 10.5), 0.7. Greenwood et al. (25-27) have used the method of Vink (28) to examine the early action of cY-amylases. In this method the average DP, as measured by the viscosity of the digest, was plotted against time. It was argued that, since a linear relationship was obtained, the degradation was random; these authors believed their argument was
FRENCH TABLE
%BVa Porcine 95 90 83 75 64
RVt" Pancreatic 16 31 40 50 53
I-Continued
RV,,c
DPpd
a-Amylase 10 19 23 27 29
160 137 76 61 58
(pH
re 10.6) 1.6 1.6 1.7 1.8 1.8 -1.7
TABLE
I
DETERMINATION OF THE DEGREE OF MULTIPLE ATTACK FROM THE RATIO OF THE TOTAL REDUCINGVALUE TOTHE REDUCINGVALUE OFTHE 67% ETHANOL POLYSACCHARIDE PRECIPITATE %BV"
RVtb
Porcine 93 90 81 72 61
RVpc
Pancreatic‘ 58 93 129 192 220
DP$
a-Amylase
Ye (pH
155 112 64 59 51
10 15 19 26 28
6.9) 5.8 6.2 6.8 7.4 7.9 7.Of
Human 99 95 93 81 68 61
Salivary
21 49 68 78 104 112
a-Amylase 7 18 21 27 35 36
(pH 6.9) 270 107 65 52 47 41
3.0 2.7 3.2 2.9 3.0 3.1 3.0
Aspergillus 97 90 80 63 51
oryzae
20 43 59 81 92
ot-Amylase 6 18 24 28 29
220 62 49 41 37
(pH
5.5) 3.3 2.4 2.5 2.9 3.2 2.9
1~ Sulfuric 97 92 89 87 81 73 58
17 27 45 70 85 91 123
17 18 22 34 38 49 48
Acid, 60’ 187 152 135 86 78 59 55
1.0 1.5 2.0 2.0 2.2 1.9 2.6 1.9
a Blue value: (At/Ao) X 100, where A0 and A, are the absorbancies (620 mr) of the iodine complex of the digest at zero time and at t minutes of hydrolysis. * Total reducing value in terms of pg of apparent maltose/ml of digest. c Reducing value of the 67yo ethanol precipi-
tate in terms of rg of apparent maltose/ml of digest. d Average degree of polymerization of the 67% ethanol precipitate determined by the quotient: [Total carbohydrate (pg/ml)/Apparent maltose WmUl X 2. B Quotient of the total reducing value divided by the reducing value of the 67% ethanol precipitate (RVJRV,). 1 Average r determination.
reinforced by their observation that acid hydrolysis also gave a linear plot. Viscosity measurements, however, are little affected by relatively low concentrations of small molecules such as maltose or maltotriose.5 In a-amyloysis, viscosity studies measure only the average DP of the resulting polymer fragments and are insensitive to the formation of low molecular weight maltodextrins by multiple attack. Thus, we interpret the viscosity studies of Greenwood et al. as indicating the random encounter6 of enzyme and substrate to form an active complex rather than the random scission characteristic of multichain action. It has been suggested (30) that the ten5 It can be shown from the viscosity data of Pulvermacher (29) that the intrinsic viscosity for maltose is only a few percentage of that for amylose (DP = 3000). 6 A process for endoamylases that is a function of diffusion, rotation, and collision of the enzyme and substrate molecules, and hence is expected to be random (22).
MULTIPLE
ATTACK
dency for multiple attack by a-amylases can be attributed to the large size of the substrate relative to the enzyme, the slow rate of diffusion of substrate and enzyme, and the cage effect of the solvent. If these were the only factors contributing to the multiple attack phenomenon, one would expect to find t,he same degree of multiple attack for enzymes of nearly equal molecular weight acting on an identical substrate under identical conditions (concentration, temperature, pH, and salts). Since PP and HS are of the same molecular size (31, 32), the differences in their degrees of multiple att’ack must be attributed to intrinsic differences in hheir enzymic properties. In addition, we have recently shown that PP shows multiple att’ack when acting on a small rapidly diffusing substrate such as cyclomaltooctaose (33). The amount of multiple attack shown by any enzyme appears to be closely related to t’he kinetic const,ants which govern the lifet’ime of the enzymesubstrate complex as compared with t,he catalytic turnover time. Thus, for a high degree of multiple attack, it is only necessary that the E-S complex lifetime be large compared with the catalytic turnover time. It is also clear that there must be a rapid rearrangement (sliding) of the substrate on t’he active site of the enzyme between successive catalytic events. The activity of PP, in terms of reducing values, at pH 10.5 is less than one-tenth that at the pH optimum (6.9). The change in action from multiple attack at pH 6.9 to multichain at pH 10.5 is most simply interpreted as a change to an unfavorable ionization &ate of one or more of the catalytic groups, thereby retarding t’hem so that they rarely function more than once during the lifetime of an effective E-S complex. On the acid side, the activity of PP at pH 4.5 is also less t)han one-tenth that at pH 6.9, yet the degree of multiple attack remains the same. Hence the low acid activity cannot be readily explained by an unfavorable ionization of the cat,alytic groups, but may be due to an unfavorabIe ionization at the binding site, or possibly t’o gross changes in protein conformat,ion so tZhat only a small fraction is enzymically active.
1’<)
BY a-AMYLASES REFERENCES
5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18.
19. 20. 21.
22. 23. 24.
25. 26.
BERNFELD, P., AND FULD, M., Helv. Chim. Ada 31, 1423 (1948). MEYER, K. H., AND GONON, W. F., Helv. Chim. Acta 34, 294 (1951). BERNFELD, P., Advan. Enzymol. 12, 379 (1951). KUNG, J. T., H.iNRaHhN, V. M., AND CALDWELL, M. L.. J. Am. Chem. Sot. 76, 5548 (1953). PAZUR, J. H., FRENCH, D., AND KNAPP, D., Proc. Iowa Ad. Sci. 67, 203 (1950). DUBE, S. K., AND NORDIN, P., Arch. Biochem. Biophys. 99, 105 (1962). ROBYT, J. F., AND FRENCH, D., Arch. Biochem. Biophys. 100, 451 (1963). W:\LKER, G. W., Biochem. J. 94, 289 (1965). HOPKINS, R. H., 14~~ JELINEK, B., Xature 166, 955 (1949). BOURNE, E. J., AITD WHELAN, W. J., Nature 166, 258 (1950). B.~ILEY, J. M., .~ND FRENCH, D., J. Biol. Chem. 226, 1 (1957). FRENCH, D., AND YOUNGQUIST, R. W., Die Stlirke 16, 425 (1963). HOFFMANN, W. S., J. Biol. Chem. 120, 51 (1937). “Technicon Autoanalyxer Methodol(1964). ogy> N-9, ” “Microglucose” ROBYT, J. F., AND WHELAN, W. J., Biochem. J. 96, 10~ (1965). MCCREADY, R. M., AND HASSID, W. Z., J. Am. Chem. Sot. 66, 1154 (1943). ROBYT, J. F., AND BEMIS, S., Anal. Biochem. 19, 56 (1967). FISCHER, ED. H., END STEIN, E. A., Biochem. Prep. 8, 27 (1961). CALDWELL, M. L., ADAMS, M., KUXG, J. T., AND TORALBALL.~, G. C., J. Am. Chem. Sot. 74, 4033 (1952). FISCHER, ED. H., AND DEMONTOMOLLIN, R., Helv. Chim. Acta 34, 1987 (1951). NELSON, N., J. Biol. Chem. 163, 375 (1944). BERNFELD, P., in “Comparative Biochemistry” (M. Florkin and H. S. Mason, eds.), Vol. III, p. 376. Academic Press, New York (1962). FRENCH, D., Bakers Digest 32, 50 (1957). BAILEY, J. M., AND WHELSN, W. J., J. Biol. Chem. 236, 969 (1961). BEMILLER, J. N., in “Starch: Chemistry and and E. F. Technology” (R. L. Whistler Paschall, eds.), Vol. I, p. 501. Academic Press, New York (1965). GREENWOOD, C. T., MACGREGOR, A. W., *ND MILNE, E. A., Die Stiirke 17, 219 (1865). GREENIVOOD, C. T., MICGREGOH, A. W., AND MILNE, E. A., Arch. Rio&em. Bioph?ys. 112, 466 (1965).
16
ROBYT
AND
27. GREENWOOD, ‘C. T., MACGREGOR, A. W., AND MILNE, E. A., Carbohydrate Res. 1, 303 (1965). 28. VINE, H., Makromol. Chem. 67, 105 (1963). 29. PULVERMACHER, O., Z. Anorg. Allegm. Chem. 113, 147 (1920). 30. PAZUR, J. H., in “Starch: Chemistry and Technology” (R.. L. Whistler and E. F. Paschall, eds.) , Vol. I, p. 142. Academic Press, New York (1965). 31. MUTZBAUER, H., AND SCHULZ, G. V., Biochim. Biophys. Acta 102, 526 (1965). 32. CILDWELL, M.L., DICKEY, E. S., H~NRAHAN,
FRENCH V. M., AND MISKO, M., 76, 143 (1954). 33. ABDULLAH, M., FRENCH, J. F., Arch. Biochem. (1966). 34. FRENCH, D., MANCUSI, J. dNr~ BRaMMER, G. L., J.
J. Am. Chem. Sot. D., AND ROBYT, Biophys. 114, 595 L., ABDULLAH, Chromatog.
M.,
1% 445
(1965).
35. FOSTER, J. F., in “Starch: Chemistry and Technology” (R. J. Whistler and E. F. PaschalIt eds.), Vol. I, p. 349. Academic Press, New York (1965).
OF
Multiple
BIOCHEMISTRY
Attack
Pancreatic,
AND
Hypothesis
Human
122, 8-16 (1967)
RIOPHYSICS
of cr-Amylase
Salivary,
JOHN F. ROBYT Department
of Biochemistry Received
and
Aspergillus DEXTER
AND
and Biophysics, February
Action:
Iowa
Action
oryzae
oc-Amyloses’
FRENCH
State University,
23,1967; accepted
of Porcine
Ames, Iowa 50010
May 5, 1967
Three conceptual action patterns of ol-amylase hydrolysis of amylose have been considered: single chain, multichain, and multiple attack. To test these concepts, curves were obtained relating the drop in amylose-iodine color to the increase in reducing value for amylolysis by human salivary (HS), porcine pancreatic (PP), Aspergillus oryzae (AO) a-amylases, and 1 M HzSO~. The observed differences in the curves for the amylases could only be interpreted as due to differences in degree of multiple attack. To test these concepts further, amylase digests at various stages of hydrolysis were separated by ethanol precipitation into polysaccharide and oligosaccharide fractions. The degree of multiple attack was determined from the ratio of the reducing value of the oligosaccharide fraction to that of the polysaccharide fraction. Under optimal conditions of pH and temperature, PP had a degree of multiple attack of 6, three times that of HS or AO. At pH 4.5, the degree of multiple attack of PP did not change, although its activity was reduced lo-fold. At pH 10.5, however, the degree of multiple attack was reduced to 0.7, approaching a multichain pattern.
It is widely believed that a-amylases from a number of sources hydrolyze starch at random points in the polymer chain to give a random distribution of products that eventually become identical (l-3). In 1953, Kung et al. (4) published a series of curves relating the drop in iodine color (blue value2) to the increase in reducing value for the action of several a-amylases upon amylose. These curves varied significantly for the individual a-amylases, thus indicat,ing that the act’ions of a-amylases were not
identical. Kung et al. (4) suggested that the differences were due to the different 01amylases hydrolyzing amylose into different chain lengths. Since then, paper chromatographic analyses have shown that, in fact, a number of or-amylases produce low molecular weight products with molecular size distributions characteristic of the individual enzymes (5-S). Chromatographic analysis of HS- and PP-amylases digests show (Fig. 1) that these two amylases produce very similar products. Therefore, their widely different blue valuereducing value curves (Fig. 2) cannot be accommodated by the explanation advanced by Kung et al. (4).
1 Journal Paper No. J-5460 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Projects 1116 and 1485. Support’ed by grants from the Corn Industries Research Foundation, NIH GM-08822 and ARS 12-14-100-l-7668 (71). Presented in preliminary form at the 150th meeting of the American Chemical Society, IXvision of Biological Chemistry (Abstracts 96C, 1965). 2 Blue value is defined as (At/A0 X 100, where A, and AL are the absorbancies (620 rnp) of the iodine complex of the digest at zero time and at t minutes of hydrolysis.
For strates
enzymic action there are three
on polymeric distinct action
subpat-
terns: single chain, multichain, and multiple attack. In the single chain action, once the enzyme forms an active enzyme-sub3 Abbreviations used: PP, porcine pancreatic a-amylase; HS, human salivary my-amylase; AO, Aspergillus oryzae or-amylase (Taka A).
8
tiuman
sal~varv
~i-Amvlase pti
6 9. 40’
Ilmu/mli C,
dqest 1OmM
of
Amvlose
11 W/ml1
chloride
porcine
Pancreatlc
(1 mu/ml) C
1OmM
digest
of Amvlose
II
mg/mlb
chloride
0
FIG. 1. Time sequence chromatographic analysis of human salivary and porcine pancreatic a-amylase action on amylose. The conditions of the digests were: amylose (1 mg/ml); 20 mM, pH 6.9 sodium glycerophosphate buffer; 10 mM NaCl; 1 mU of enzyme/ml. Aliquots (100 ~1) were placed on the chromatogram. The first two columns represent cont.rols; thereafter a time sequence in minutes and hours. The chromatograms were obtained by the techniques of French el al. (34).
strate (E-S) complex, it catalyzes reaction in a “zipper” fashion toward one end of the chain and does not form an active complex with another substrate, until it comes to the end of the first chain.4 The multichain process is the classical random action in which the enzyme catalyzes the hydrolysis of only one bond per effective encounter. In the multiple-attack act’ion, once the enzyme forms an enzyme-polymer complex, in which there is suitable geometry for catalysis, the enzyme may catalyze the hydrolysis of several bonds before it dissociates and forms a new active complex with another polymer chain. The cleqreeof mult’iple attack may be defined as the average number of catalytic events, following the first, during t’he lifetime of an individual E-S complex. The single chain and multichain mechanisms 4 There is the possibility of more than one active site per enzyme molecule. In this case, the enzyme could form a complex with another substrate molecule at the second active site. However, if the two active sites were independent, it would be assumed that the individual active site would not form a complex with a second polymer until it had come to the end of the first chain.
represent the two extreme cases of polymer degradation; the multiple-attack mechanism is a more general concept including multichain and single chain mechanisms a,s extreme special cases. Up unt’il now, these concepts have been applied primarily to exoenzymes, e.g., pamylase and phosphorylase (9, 10). It has been shown that p-amylase has a multiple attack action (11, 12). a-Amylases, by contrast’, are generally regarded as being endoenzymes, t,hat is, enzymes that initiate attack at, random interior positions along the polymer chain. It is obvious that the gross facets of action pattern must be different for exo- and endo-enzymes. However, if one examines the details of action, it is clear t’hat the three concepts, originally developed for exo-enzymes, may a’lso be applied to the action of endo-enzymes (cf. Fig. 3). In the present investigation, we have devised an experimental method for measuring the degree of multiple attack, and have shown that various or-amylases exhibit varying degrees of multiple attack. Also, we have shown that t’he degree of multiple
10
ROBYT
60
AND
FRENCH
-
BLUE VALUE
30 -
40
-
30
-
20
-
P P (p H 4.5 I’\\
IO -
0
2
4
6
9
REDUdlNG
IO
I2
14
VALUE
I6
I6
AS
20
22
24
% APPARENT
26
28
30
32
34
MALTOSE
FIG. 2. Comparison of the drop in iodine color (blue value) with the increase in reducing value for the hydrolysis of amylose by various catalysts. Blue value is defined as (A JA,) X 100, where L4a and A, are the absorbancies (620 rnp) of the iodine complex of the digest at zero time and at t minutes of hydrolysis. (0) PP (pH 6.9), porcine pancreatic a-amylase at pH 6.9, 10 mM Cl-, and 40”; (0) HS, human salivary a-amylase at pH 6.9, 10 mM Cl-, and 40”; (n) PP (pH 4.5), porcine pancreat.ic ol-amylase at pH 4.5, 10 mM Cl-, and 40”; (A) AO, 4spergiZZu.s oryzae a-amylase at pH 5.5, and 40”; (A) 1 M sulfuric acid at 60”; (0) PP (pH 10.5) porcine pancreatic ol-amylase at pH 10.5, 10 mM Cl-, and 40”.
attack depends on t$he reaction conditions. For, example, with PP under optimal conditions (pH 6.9), the degree of multiple attack is about 6; at pH 10.5, the degree of
multiple attack drops to essentially zero, which corresponds to the multichain mechanism. With HS under opt,imal conditions, the degree of multiple att,ack is only about 3;
MULTIPLE
A
SINGLE
B
MULTIPLE
ATTACK
BY wAMYLASES
CHAIN
ATTACK
C. MULTI CHAIN
E,
FIG. 3. Types of attack action patterns for endo amylases. Three cases are presented; each case illustrates the action of a single enzyme molecule. The arrows represent the catalytic hydrolysis of a glycosidic bond; 0 represents an amylose molecule made up of glucosyl units linked 01, 1 + 4; + represents a reducing hemiacetal end group. The numbers refer t,o the sequence of hydroIytic events by the enzyme. The oligosaccharide product specificity in cases A and B is arbitrarily assumed to be maltose and maltotriose. The authors have assumed a definite polarity of the enzyme action toward the reducing end. However, the actual direction of the action is not known; it may as well be toward the nonreducing end or the enzyme may not have a preference, although the authors are inclined to believe that the action is in one direction or the other. For illustrative purposes the amylose molecule however, it undoubtedly possesses a certain amount of is pictured as a long “string”; secondary helical structure (35) that is not illustrated.
t,herefore, we int,erpret t)he blue value-reducing value curve differences as reflecting primarily differences in the degree of multiple attack by t*hese otherwise very similar a-amylases. EXPERIMENTAL
PROCEDURE
Analytical meihods. Reducing values were measured by the alkaline ferricyanide-cyanide procedure (13) with the Technicon Autoanalyzer. The method was not affected by the chain length of the maltodextrins (14) since equal reducing values were obt,ained by equimolar amounts of and maltotriose. This result glucose, maltose,
indicates that the method is a stoichiometric measure of the hemiacetal groups (reducing groups) present in a maltodextrin sample. In the present work, it has been assumed that themethod will similarly determine the hemiacetal groups for higher saccharides, e.g., 4-1000 glucose units. The blue value was determined by adapting the conditions of McCready and Hassid (15) for use wit,h the Technicon AutoanalyJer (16). Total carbohydrate was determined by t,he phenolsulfuric acid procedure adapted for use with the Technicon Autoanalyzer (16). Substrate. Superlose, a commercial amylose from Stein-Hall & Co., New York, was recrystallized twice with 1-butanol; 50 gm of Superlose
12
ROBYT
AND
was suspended in 500 ml of dimethyl sulfoxide and stirred until dissolved (about 10 hours). The dimethyl sulfoxide solution was diluted with 4.5 liters of water, and 200 ml of I-but,anol was added. The mixture was stirred overnight and centrifuged. The butanol-amylose complex was redissolved by adding small amounts to 5 liters of boiling water. After the complex was dissolved, and the solution had cooled to about 65”, 200 ml of 1-butanol was added, and the mixture was stirred overnight. The butanol-amylose complex was recovered by centrifugation and successively treated five times with 250 ml of anhydrous acetone. The average degree of polymerization (DP) of the recrystallized amylose, as determined by reducing value and phenol-sulfuric acid analysis, was 1000 f 50. Enzymes. Human salivary a-amylase (HS) was crystallized according to the method of Fischer and Stein (17). Porcine pancreatic cu-amylase (PP), crystallized twice by the method of Caldwell et al. (18), was obtained from Worthington Biochemicals, Freehold, New Jersey. Aspergillus oryzae a-amylase (AO) was crystallized twice according to the method of Fischer and DeMontmollin (19). Enzyme activity was measured by determination of reducing values using the ferricyanide method (13) or the Nelson copper method (20). Activity is expressed in International Units, viz., one unit (U) is that amount of enzyme that will hydrolyze one pmole of bond per minute at 40” and the optimal pH of the enzyme. Digests. Enzymatic digests. Amylose (124 mg) was dissolved in 2 ml of dimethyl sulfoxide (25”; about 30 minutes); this was diluted with 12 ml of 200 rnM buffer (pH 6.9 and 5.5, sodium glycerophosphate; pH 10.5, sodium glycinate; pH 4.5, acetate), 12 ml of 100 mM sodium chloride, and diluted to 120 ml with water. Enzyme was added to initiate the reaction: 300 mU at the optimal pH values (6.9 or 5.5), and 6.0 U at unfavorably low or high pH (4.5 and 10.5). The digests were incubated at 40”. Duplicate 5-ml aliquots were taken at various times over 120 minutes and added to 0.1 of 5 M trichloroacetic acid (for digests at pH 10.5,0.2 ml of acid was used). After standing approximately 30 minutes, the samples were neutralized with 5 M sodium hydroxide to pH 6 (pH paper). Acid digests. Amylose (210 mg) was dissolved in 2 ml of dimethyl sulfoxide and diluted to 100 ml with water; after equilibration at BO”, 50 ml of 3 M sulfuric acid was added; at various intervals 5-ml aliquots were taken in pairs, placed in an ice bath, and neutralized with 2 ml of 5 M sodium hydroxide to pH 6 (pH paper). Bernfeld (21) has suggested that retrogradation of amylose might be the cause for the differences observed for the blue value-reducing value curves
FRENCH of Kung et al. (4). With the concentration of amylose used in our digests (1 mg/ml), the formation of insoluble precipitates was never observed, and the carbohydrate analysis for each aliquot remained constant. In addition, identical curves were obtained by using different concentrations of amylase with a consequent sampling at different 1engt)hs of time. Since retrogradation is a function of substrate concentration and time, it would be impossible to get reproducible curves if retrogradation were taking place during amylolysis. Fractionation of enzyme digests by ethanol precipilation. Two volumes of anhydrous ethanol were added to one of the two 5-ml aliquots taken at various times. The samples were placed at 5” overnight. The resulting precipitates were centrifuged and washed twice with 5 ml of 70% ethanol and once with 5 ml of anydrous ethanol. The precipitate was then dissolved in 0.2 ml of dimethyl sulfoxide and diluted with 5 ml of water. The ethanol precipitates did not contain any small maltodextrins in the range of glucose to maltododecaose as judged by paper chromatographic analysis. Under the conditions of the experiment, the smallest polymer that could be precipitated had an average degree of polymerization of 20 glucose units. The reducing value and the carbohydrate COIIt,ent were determined for each ethanol-precipitated sample, from which the average DP could be calculated. The reducing value, blue value, and carbohydrate content were determined for each of the other aliquots that were not treated with ethanol. The degree of multiple attack (Table I) was calculated from the experimental data, as explained in the Discussion. DISCUSSION
The blue value versus reducing value curves (Fig. 2) show that for an equal drop in blue value (e.g., 60%) the three ar-a’mylases PP, HS, and A0 have different percentage conversions to apparent’ maltose when operating under optimum conditions. These differences in reducing values may be interpreted as differences in the degree of multiple attack as follows. Let us imagine that the intial substrate contains 1000 glucose units and that a certain enzyme encounters the substrate in the center of the chain and cleaves it into two fragments of 500 units each. If five additional glucosidic bonds are broken from one of the fragments to give five maltose molecules, the polymer fragment would be left with 490 units. If, with a different enzyme, ten bonds are
MULTIPLE
ATTACK
broken to give ten molecules of maltose, the polymer fragment would have 450 units remaining. The total blue value of t’he 500 + 480 fragments would be very nearly the same as that of the 500 + 490 fragments (23). However, t,he reducing value of t’he digest forming t,en maltose molecules would be almost t,wice that of the digest forming five maltose molecules. Thus, differences in reducing values for two enzyme digest,s having identical blue values point’ st’rongly to differences in the degree of multiple attack. The argument is lit)tle affected if maltotriose or maltotetraose result inst’ead of maltose. The data of Fig. 2, therefore, show qualitatively that the degree of multiple &tack decreases in the following order: PP (pH 6.9 and 4.5), HS, AO, and PI’ (pH 10.5). Acid hydrolysis, by its very nature, should give a degree of multiple attack of zero. Hence one might expect that it would show the lowest reducing value for an equivalent’ drop in blue value. In fact, however, the &ion of PI’ at pH 10.5 (Fig. 2) shows a lower reducing value than acid hydrolysis at equal blue values, with the differences becoming more marked as the reactions proceed. These differences must reflect (a) t#he avoidance of chain ends by PP, so that no glucose is produced, and (b) the preferent,ial attack by acid on the terminal linkages, thus producing somewhat, more glucose than in a purely random process (24). These “end-effects” are of litt#le significance during t,he early stages of the hydrolysis when the average size of the polymer molecules is large. The initial coincidence of t,lre pH 10.5 PI’ curve with that of acid hydrolysis is compatible wit,h the idea that t,he i&al a&& by a-amglase is at’ a random position along t’he polymer chain. On the other hand, after extensive hydrolysis, end-effects substantially modify the action pnt,tern and finally determine the various final product dist,rlbuOions found with va.rious cr-amylnses. The rationale behind the calculat’ion of t,he degree of multiple attack from the ethanol fractionatjion experiment is as follows. The tot’al increase in reducing power represents the sum of two types of bond cleavages:
BY wAMYLASES
13
(a) primary attack on a polyasccharide molecule to give two macromolecular (polysaccharide) fragments, and (b) secondary attack on t’he newly formed end of one polymer fragment t’o give low molecular weight oligosaccharides. The polymers may be easily separated from the oligosaccharides by ethanol precipitation. Kew reducing groups in the polysaccharide fraction are equal in number to the number of effective encounters. New reducing groups in the oligosaccharide fraction are equal in number to the number of bonds broken in secondary phases of multiple attack. The quotient T of the total reducing value, which is a measure of t,he total number of bonds broken, divided by the reducing value of the ethanol precipitate, which is a measure of the number of effective encounters, gives t’he number of bonds broken per effective encounter. Since the first bond broken releases a polymer fragment,, the average number of subsequen~t bonds broken (~-1) is numerically equal to the degree of multiple attack. The experimentally determined value, T, remained relatively constant until the blue value had dropped to around 50 74. Only samples whose blue value had not dropped below 50 C/owere used in t,he analyses, The increase in 1’ in the later stages of the reacGon is expected because of the formation of small polymers that arc: not, completely precipitated by C,S% ethanol. In this analysis it must be assumed t,hat formation of oligosaccharides through primary attack is insignificant. This is obviously a better assumption in the initial phases of hhe react,ion when the DP of the subst,rate is many hundreds or t,housands. In the later stages of the reaction, when t,he average DP of the polymeric substrate is less t,han 30-40, the assumption breaks down altogether. The results of the ethanol fract)ionation experiments (Table I) give a quantitative index for the number of attacks occurring per effective encounter. The data confirm t’he interpretation of the blue value-reducing value curves (Fig. 2). For PP, the degree of mult’iple attack (~-1) is A; that is, for every effective encount’er, PP on the average breaks six bonds and releases maltose, maltot,riose, or malt,ot,etraose. For HS, the
14
ROBYT
AND
degree of multiple attack is 2; for AO, 1.7; and for PP (pH 10.5), 0.7. Greenwood et al. (25-27) have used the method of Vink (28) to examine the early action of cY-amylases. In this method the average DP, as measured by the viscosity of the digest, was plotted against time. It was argued that, since a linear relationship was obtained, the degradation was random; these authors believed their argument was
FRENCH TABLE
%BVa Porcine 95 90 83 75 64
RVt" Pancreatic 16 31 40 50 53
I-Continued
RV,,c
DPpd
a-Amylase 10 19 23 27 29
160 137 76 61 58
(pH
re 10.6) 1.6 1.6 1.7 1.8 1.8 -1.7
TABLE
I
DETERMINATION OF THE DEGREE OF MULTIPLE ATTACK FROM THE RATIO OF THE TOTAL REDUCINGVALUE TOTHE REDUCINGVALUE OFTHE 67% ETHANOL POLYSACCHARIDE PRECIPITATE %BV"
RVtb
Porcine 93 90 81 72 61
RVpc
Pancreatic‘ 58 93 129 192 220
DP$
a-Amylase
Ye (pH
155 112 64 59 51
10 15 19 26 28
6.9) 5.8 6.2 6.8 7.4 7.9 7.Of
Human 99 95 93 81 68 61
Salivary
21 49 68 78 104 112
a-Amylase 7 18 21 27 35 36
(pH 6.9) 270 107 65 52 47 41
3.0 2.7 3.2 2.9 3.0 3.1 3.0
Aspergillus 97 90 80 63 51
oryzae
20 43 59 81 92
ot-Amylase 6 18 24 28 29
220 62 49 41 37
(pH
5.5) 3.3 2.4 2.5 2.9 3.2 2.9
1~ Sulfuric 97 92 89 87 81 73 58
17 27 45 70 85 91 123
17 18 22 34 38 49 48
Acid, 60’ 187 152 135 86 78 59 55
1.0 1.5 2.0 2.0 2.2 1.9 2.6 1.9
a Blue value: (At/Ao) X 100, where A0 and A, are the absorbancies (620 mr) of the iodine complex of the digest at zero time and at t minutes of hydrolysis. * Total reducing value in terms of pg of apparent maltose/ml of digest. c Reducing value of the 67yo ethanol precipi-
tate in terms of rg of apparent maltose/ml of digest. d Average degree of polymerization of the 67% ethanol precipitate determined by the quotient: [Total carbohydrate (pg/ml)/Apparent maltose WmUl X 2. B Quotient of the total reducing value divided by the reducing value of the 67% ethanol precipitate (RVJRV,). 1 Average r determination.
reinforced by their observation that acid hydrolysis also gave a linear plot. Viscosity measurements, however, are little affected by relatively low concentrations of small molecules such as maltose or maltotriose.5 In a-amyloysis, viscosity studies measure only the average DP of the resulting polymer fragments and are insensitive to the formation of low molecular weight maltodextrins by multiple attack. Thus, we interpret the viscosity studies of Greenwood et al. as indicating the random encounter6 of enzyme and substrate to form an active complex rather than the random scission characteristic of multichain action. It has been suggested (30) that the ten5 It can be shown from the viscosity data of Pulvermacher (29) that the intrinsic viscosity for maltose is only a few percentage of that for amylose (DP = 3000). 6 A process for endoamylases that is a function of diffusion, rotation, and collision of the enzyme and substrate molecules, and hence is expected to be random (22).
MULTIPLE
ATTACK
dency for multiple attack by a-amylases can be attributed to the large size of the substrate relative to the enzyme, the slow rate of diffusion of substrate and enzyme, and the cage effect of the solvent. If these were the only factors contributing to the multiple attack phenomenon, one would expect to find t,he same degree of multiple attack for enzymes of nearly equal molecular weight acting on an identical substrate under identical conditions (concentration, temperature, pH, and salts). Since PP and HS are of the same molecular size (31, 32), the differences in their degrees of multiple att’ack must be attributed to intrinsic differences in hheir enzymic properties. In addition, we have recently shown that PP shows multiple att’ack when acting on a small rapidly diffusing substrate such as cyclomaltooctaose (33). The amount of multiple attack shown by any enzyme appears to be closely related to t’he kinetic const,ants which govern the lifet’ime of the enzymesubstrate complex as compared with t,he catalytic turnover time. Thus, for a high degree of multiple attack, it is only necessary that the E-S complex lifetime be large compared with the catalytic turnover time. It is also clear that there must be a rapid rearrangement (sliding) of the substrate on t’he active site of the enzyme between successive catalytic events. The activity of PP, in terms of reducing values, at pH 10.5 is less than one-tenth that at the pH optimum (6.9). The change in action from multiple attack at pH 6.9 to multichain at pH 10.5 is most simply interpreted as a change to an unfavorable ionization &ate of one or more of the catalytic groups, thereby retarding t’hem so that they rarely function more than once during the lifetime of an effective E-S complex. On the acid side, the activity of PP at pH 4.5 is also less t)han one-tenth that at pH 6.9, yet the degree of multiple attack remains the same. Hence the low acid activity cannot be readily explained by an unfavorable ionization of the cat,alytic groups, but may be due to an unfavorabIe ionization at the binding site, or possibly t’o gross changes in protein conformat,ion so tZhat only a small fraction is enzymically active.
1’<)
BY a-AMYLASES REFERENCES
5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18.
19. 20. 21.
22. 23. 24.
25. 26.
BERNFELD, P., AND FULD, M., Helv. Chim. Ada 31, 1423 (1948). MEYER, K. H., AND GONON, W. F., Helv. Chim. Acta 34, 294 (1951). BERNFELD, P., Advan. Enzymol. 12, 379 (1951). KUNG, J. T., H.iNRaHhN, V. M., AND CALDWELL, M. L.. J. Am. Chem. Sot. 76, 5548 (1953). PAZUR, J. H., FRENCH, D., AND KNAPP, D., Proc. Iowa Ad. Sci. 67, 203 (1950). DUBE, S. K., AND NORDIN, P., Arch. Biochem. Biophys. 99, 105 (1962). ROBYT, J. F., AND FRENCH, D., Arch. Biochem. Biophys. 100, 451 (1963). W:\LKER, G. W., Biochem. J. 94, 289 (1965). HOPKINS, R. H., 14~~ JELINEK, B., Xature 166, 955 (1949). BOURNE, E. J., AITD WHELAN, W. J., Nature 166, 258 (1950). B.~ILEY, J. M., .~ND FRENCH, D., J. Biol. Chem. 226, 1 (1957). FRENCH, D., AND YOUNGQUIST, R. W., Die Stlirke 16, 425 (1963). HOFFMANN, W. S., J. Biol. Chem. 120, 51 (1937). “Technicon Autoanalyxer Methodol(1964). ogy> N-9, ” “Microglucose” ROBYT, J. F., AND WHELAN, W. J., Biochem. J. 96, 10~ (1965). MCCREADY, R. M., AND HASSID, W. Z., J. Am. Chem. Sot. 66, 1154 (1943). ROBYT, J. F., AND BEMIS, S., Anal. Biochem. 19, 56 (1967). FISCHER, ED. H., END STEIN, E. A., Biochem. Prep. 8, 27 (1961). CALDWELL, M. L., ADAMS, M., KUXG, J. T., AND TORALBALL.~, G. C., J. Am. Chem. Sot. 74, 4033 (1952). FISCHER, ED. H., AND DEMONTOMOLLIN, R., Helv. Chim. Acta 34, 1987 (1951). NELSON, N., J. Biol. Chem. 163, 375 (1944). BERNFELD, P., in “Comparative Biochemistry” (M. Florkin and H. S. Mason, eds.), Vol. III, p. 376. Academic Press, New York (1962). FRENCH, D., Bakers Digest 32, 50 (1957). BAILEY, J. M., AND WHELSN, W. J., J. Biol. Chem. 236, 969 (1961). BEMILLER, J. N., in “Starch: Chemistry and and E. F. Technology” (R. L. Whistler Paschall, eds.), Vol. I, p. 501. Academic Press, New York (1965). GREENWOOD, C. T., MACGREGOR, A. W., *ND MILNE, E. A., Die Stiirke 17, 219 (1865). GREENIVOOD, C. T., MICGREGOH, A. W., AND MILNE, E. A., Arch. Rio&em. Bioph?ys. 112, 466 (1965).
16
ROBYT
AND
27. GREENWOOD, ‘C. T., MACGREGOR, A. W., AND MILNE, E. A., Carbohydrate Res. 1, 303 (1965). 28. VINE, H., Makromol. Chem. 67, 105 (1963). 29. PULVERMACHER, O., Z. Anorg. Allegm. Chem. 113, 147 (1920). 30. PAZUR, J. H., in “Starch: Chemistry and Technology” (R.. L. Whistler and E. F. Paschall, eds.) , Vol. I, p. 142. Academic Press, New York (1965). 31. MUTZBAUER, H., AND SCHULZ, G. V., Biochim. Biophys. Acta 102, 526 (1965). 32. CILDWELL, M.L., DICKEY, E. S., H~NRAHAN,
FRENCH V. M., AND MISKO, M., 76, 143 (1954). 33. ABDULLAH, M., FRENCH, J. F., Arch. Biochem. (1966). 34. FRENCH, D., MANCUSI, J. dNr~ BRaMMER, G. L., J.
J. Am. Chem. Sot. D., AND ROBYT, Biophys. 114, 595 L., ABDULLAH, Chromatog.
M.,
1% 445
(1965).
35. FOSTER, J. F., in “Starch: Chemistry and Technology” (R. J. Whistler and E. F. PaschalIt eds.), Vol. I, p. 349. Academic Press, New York (1965).