ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 198, No. 2, December, pp. 627-631, 1979

The Mechanism

of Q-Enzyme Action and Its Influence Structure of Amylopectin1,2

on the

DOV BOROVSKY,” ERIC E. SMITH,* WILLIAM J. WHELAN,*,3 DEXTER FRENCH,? AND SHOICHI KIKUMOTOt *Depcrtment of Biochemistry, University of Miami School and TDepartment of Biochemistry and Biophysics,

of Medicine, P.O. Box 016129, Miami, Florida 33101, Iowa State University, Ames, Iowa, 50011

Received August 17, 1979 Q-Enzyme is responsible for the synthesis of the 1,6-branch linkages in amylopectin. Its action on a model amylodextrin containing a single branch linkage has been studied. It is concluded that the enzymic process whereby the branch linkages of amylopectin are synthesized is a random action of the branching enzyme on a complex-possibly a double helixformed between two 1.4~a-ducan chains. This action pattern predicts a novel arrangement of the units chains in amylopectin.

Q-Enzyme (EC 2.4.1.18) is responsible for the synthesis of the 1,6-branch linkages of the starch component, amylopectin. Based on experiments in which potato Q-enzyme was allowed to synthesize 1,6-branch linkages in amylose or growing amylose chains, we adduced evidence (l-3) that the substrate for branch formation by Q-enzyme is a complex formed between two chains, that the act of branching is a transglycosylation in which one chain is split and a portion of it is joined to the second chain (interchain transfer), and that the formation of a covalent branch linkage between the two chains stabilizes their interaction and accelerates the introduction of further branch linkages. We now have a model low-molecular weight substrate for the enzyme that has permitted us to test these conclusions directly and we draw attention to the implications of the proposed reaction mechanism of Q-enzyme for the structure of amylopectin. MATERIALS

AND METHODS

Potato Q-enzyme was prepared and assayed as by Borovsky et al. (2). It is an electrophoretically homog* Dedicated to Professor Frank Dickens, on the occasion of his eightieth birthday. ’ Supported by grants from the United States Public Health Service, National Institutes of Health (AM 12352 to W.J.W. and GM 08822 to D.F.). 3 To whom correspondence should be addressed. 627

enous protein free from a-amylase (EC 3.2.1.1), D-enzyme (EC 2.4.1.25), pullulanase (EC 3.2.1.41), and phosphorylase (EC 2.4.1.1). Pullulanase was prepared and assayed as by Mercier et al. (4). Isoamylase (EC 3.2.1.68) was prepared as by GunjaSmith et al. (5). Amylodextrin (see below) was labeled with NaB3H, (200 mCi/nmol, New England Nuclear Corporation), as by Richards and Whelan (6). Radioactivity was measured in a Packard TriCarb Scintillation Spectrometer. Solutions containing 3H (0.2 ml) were applied to 12-mm squares of Whatman 31ET paper. The paper was dried under an infrared lamp and placed in a vial containing 10 ml of scintillation solution (PP0,4 5 g, POPOP, 0.5 g, toluene, 1 liter). Amylodextrin mixtures were fractionated on a BioGel P-6 column (94 x 2.5 cm) that was previously calibrated with linear maltosaccharides of known average degree of polymerization (DP). Column fractions were assayed with amyloglucosidase-glucose oxidase (7) or with phenol-sulfuric acid (8). The total amount of carbohydrate applied to a column was usually 4.0 mg. In some experiments this was not possible. Nevertheless, in order to make for easier comparison, the data in the figures have been depicted as if each experiment was based on 4.0 mg of carbohydrate. The conditions of treatment of the amylodextrin with enzymes or with borate were as follows: Q-enzyme, 13.5 mIU/ml in 100 mM sodium citrate, pH 7.0, for 24 h at 24°C; isoamylase, 2.6 IU/ml in 100 mM sodium citrate, pH 7.0 at 37°C for 24 h; pullulanase, 1.2 IUlml in 100 mM sodium citrate, pH 7.0 at 37°C for 24 h, borate, * Abbreviations used: DP, degree of polymerization; POP, polyphenylene oxide; POPPO, p-bis[2-(5phenyloxazolyl)]-benzene. 0003-9861/79/140627-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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20 mM sodium borate, pH 10.0 at 65°C for 24 h. Treatments with P-amylase (EC 3.2.1.2) were conducted as by Borovsky et al. (2). The experiments with Q-enzyme were carried out with an amylodextrin substrate derived from waxymaize-starch granules by treatment with 16% sulfuric acid (w/v) at 38°C for 43 days, followed by separation of the residual insoluble polysaccharide into three fractions (I-III) (9-11). The amylodextrin (fraction II) consists essentially of two 1,4-ru-glucan chains joined by a l&branch linkage. One of these, the A chain [in the terminology of Peat et al. (12); see also Fig. 31, contains 11-12 glucose residues while the second chain (C), carrying the free reducing end group, is 13- 14 glucose units long with the point of branching lying 2-3 glucose residues from the reducing end (10). The evidence for this description is as follows. The amylodextrin was converted almost quantitatively into glucose by fungal amyloglucosidase (7). The degree of conversion into maltose by P-amylase was 81% and by P-amylase and pullulanase acting together was 164%. These observations indicate that the oligosaccharide is a 1,4-cu-glucan (as evidenced by the hydrolysis by amyloglucosidase and by P-amylase), containing a 1,6-branch linkage or linkages (as evidenced by the hydrolysis to completion by P-amylase plus pullulanase). The relative degrees of hydrolysis by P-amylase versus P-amylase plus pullulanase indicate that most or all of the amylodextrin molecules contained only one branch linkage and also permit the calculation (see Ref. (13)) of the above-stated average position of the branch linkage relative to the reducing end of the C chain. In confirmation of these conclusions was the fact that paper chromatography of the /3-amylolysate of the dextrin showed the presence of maltose and oligosaccharides ranging in DP from 6 to 9, these being in the size range expected for P-amylase limit dextrins derived from an amylodextrin of the average structure described. The amylodextrin, shown diagrammatically in Fig. 3, is therefore a very simple prototype of an amylopectin molecule, ideal as a model substrate for Qenzyme action. Its behavior on a molecule sieve is shown in Fig. 1. RESULTS

AND DISCUSSION

Based on our hypothesis (l-3) of the nature of the substrate for Q-enzyme and the mechanism of the enzyme action, it could be predicted that although the two chains making up the amylodextrin molecule were by themselves too short to act as substrates for Q-enzyme, the fact of their being covalently linked through a 1,6-bond might confer sufficient stability on any complex that they could form with each other, as

ET AL.

to render the complex susceptible to branching enzyme action. This prediction was verified by an examination of the component 1,4-a-glucan chains in the dextrin before and after it had been treated with Q-enzyme. The component chains were released from the dextrin by treatment with a debranching enzyme (pullulanase and/or isoamylase) specific for the hydrolysis of the 1,6-branch linkage. Figure 1 shows the carbohydrate profile of the original amylodextrin on a molecular sieve, as a function of DP. The average DP was 25. Treatment with the debranching enzymes released products with average DP 10-15, as expected from the structure deduced for the amylodextrin (see above). We will return later to the fact that the debranching, under the conditions shown, was incomplete. When the amylodextrin was treated with Q-enzyme prior to being debranched, the chains of lowest DP set free after debranching (Fig. 2) were significantly shorter (DP s 5) than those seen before the amylodextrin had been treated with Q-enzyme (Fig. 1). A second sequential treatment with Qenzyme and a mixture of debranching enzymes brought about a still greater change in the overall carbohydrate profile. The chains of DP G 5 were now the most prominent species present. The fate of the C chains of the amylodextrin, containing the single reducing chain end, could be seen by first incorporating 3H into the reducing end with labeled borohydride and then scanning the carbohydrate chains produced by the enzyme actions for their content of tritium (Fig. 2). As expected, the linear chains set free by debranching enzyme from the original amylodextrin (Fig. 1) were not substrates for Q-enzyme. No change in size of these chains was noted when they were successively incubated with branching and debranching enzymes (experiment not shown). A comparison of the products of direct debranching of the amylodextrin (Fig. 1) and after one cycle of branching and debranching (Fig. 2) reveals that it was the branching action that created the 1,4-aglucan chains with DP s 5. As could be predicted from the release of these short

Q-ENZYME

ACTION

AND AMYLOPECTIN

chains from molecules with average DP of 25, the average DP of the other carbohydrate species in the mixture lay between 15 and 20 (Fig. 2). This latter material clearly still contained branch linkages and, like the original amylodextrin, proved capable of being further branched (and debranched) in a second cycle of treatments with Q-enzyme and isoamylase (Fig. 2). These observations can be interpreted in terms of Fig. 3. Q-Enzyme is depicted as being able to cleave the A chain of the amylodextrin and thereby to place a second branch linkage on the C chain. There are now two A chains in the product, shown as A’ and A”. In the same way, scission of the C chain to place a new A chain (A’) onto the original A chain is also shown. The original A chain then becomes a B chain (12). Branching in the latter direction (C + A) would result in the C chain being shortened and therefore after the branchingldebranching sequence the tritium marker should appear in smaller chains. Figure 2 shows that this happened. The short chains formed by such branching would have a higher specific activity than the original amylodextrin. Branching in the direction A + C

200

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3w ELUTION 30

20

450 350 400 VOLUME lmli

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500

1

DP

FIG. 1. The behavior of the amylodextrin sieve (Bio-Gel P-6) before and after debranching. The carbohydrate profile of the untreated amylodextrin is shown as (- - - ). After treatment with isoamylase the carbohydrate profile became (- - -), and after successive treatments with borate and isoamylase was (-). The greatest extent of debranching was achieved by successive treatments with borate, isoamylase, and isoamylase plus pullulanase (-).

STRUCTURE

200

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300 ELUTION 25 I! I 30 20

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350 400 650 500 VOLUME (hII I5 5 : : : IDP I 10

FIG. 2. The introduction of branches into amylodextrin by treatment with Q-enzyme as revealed by the profile of unit chains seen on debranching the product and fractionating on a molecular sieve (Bio-Gel P-6). The amylodextrin was labeled at the reducing end with 3H. Its carbohydrate (-) and tritium profiles (- - -) are shown in diagram a. Diagram b illustrates the fact that after one cycle of sequential treatments with Q-enzyme, and isoamylase plus pullulanase, the carbohydrate profile became that shown by (-) and the tritium by (- - -). Another portion of (unreduced) amylodextrin was similarly sequentially treated with Q-enzyme, borate, and isoamylase, when its carbohydrate profile also became that depicted by (-), but changed to (-. -) after a second cycle of sequential treatments with Q-enzyme and isoamylase.

would give short chains of lower specific activity, as observed (Fig. 2). However, debranching of the amylodextrin was incomplete, rendering inconclusive any deductions regarding the extents of branching in each direction. It is relevant to record that the amylodextrin was exceptionally resistant to the actions of the debranching enzymes. Isoamylase, when used in amounts and for a duration that would result in the complete debranching of glycogen, only partly debranched the amylodextrin (Fig. 1). In order to achieve more substantial debranching we had resort to repeated debranching treatments, to the use of a mixture of isoamylase

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BOROVSKY

Al

c lo1

lb1

FIG. 3. Postulated modes of action of Q-enzyme on the amylodextrin. The A, B, C symbolism for the 1,4-a-glucan chains is as in Ref. (12). A 1,6-branch linkage is denoted as &. R is the reducing end of the C chain. Product (a) is formed by transglycosylation from chain A (donor) to C (acceptor). Product (b) is formed by transglycosylation from C (donor) to A (acceptor).

and pullulanase, and to preincubation of the dextrin in borate at pH 10 and 65°C overnight, a treatment that our colleague, Dr. G. Wober, discovered is effective in helping pullulanase completely to debranch amylopectin (unpublished observation). This resistance of the amylodextrin to debranching suggests that there are associative forces between the two chains held together by the branch link. One of us (10) pointed out some years ago that molecular models of the helical 1,4-a-glucan chains can be assembled into compact, close-fitting double helices. It could be that a double helix conformation is the substrate for the branching enzyme but resists action of a debranching enzyme. 5 This concept would 5 We have already reported the inability of pullulanase to effect complete debranching of amylopectin (14) and a complete inability of the enzyme to debranch liver glycogen (15). Our conclusion had been that “the ability or otherwise of pullulanase to attack a polysaccharide must be determined by the overall tertiary structure of the polymer” (16). But now it is evident that the same impedance to debranching can reside in a simple amylodextrin, containing only a single branch linkage. The tentative conclusion may be drawn that it is the degree of association (double helix formation?) between any two chains joined by a branch linkage that determines the susceptibility of the branch linkage to hydrolysis. This is consistent with our finding that if fl-amylase is allowed to act on amylopectin or glycogen in conjunction with pullulanase then the total hydrolysis of the l&bonds ensues (16).

ET AL.

allow one to depict the action of branching enzyme during amylopectin synthesis as in Fig. 4. The enzyme is envisaged as acting at random on two chains, long enough to form a stable complex, with branches being formed in either direction. This would lead to an arrangement of A and B chains, as shown at the bottom of Fig. 4, that is compatible with many of the already known characteristics of the relative arrangement of these chains in amylopectin (17), and has the advantage that it incorporates a knowledge of the action pattern of the branching enzyme. This model for branching action also overcomes what has previously been a conceptually difficult fact to understand, namely that the branching enzyme will apparently transglycosylate chain fragments that are themselves already branched (18). The model for branching action shown in Fig. 4 makes no distinction between what would be regarded as the transglycosylation of a linear fragment of chain or of a branched fragment. Experimental evidence for the suggestion that 1,4-a-glucan chains can form double helices has now been advanced by Wu and Sarko as a result of X-ray diffraction studies of crystalline potato B-amylose (19) and A-amylose (20).

FIG. 4. Depicting the action of Q-enzyme (branching enzyme) on two 1,4-a-glucan chains lined in parallel association (double helix). The chains are assumed to lie with their reducing ends in the same direction. The enzyme introduces branch points ( J ) at random; either chain can act as donor or acceptor. The product is shown in helical form, and in an exploded form in the style used to depict the laminated amylopectin structure of Haworth et al. (12).

Q-ENZYME

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AND

AMYLOPECTIN

REFERENCES 1. BOROVSKY, D., SMITH, E. E., AND WHELAN, W. J. (19’75) FEBS Lett. 54, 201-205. 2. BOROVSKY, D., SMITH, E. E., AND WHELAN, W. J. (1975) Eur. J. Biochem. 59, 615-625. 3. BOROVSKY, D., SMITH, E. E., AND WHELAN, W. J. (1976) Eur. J. Biochem. 62, 307-312. 4. MERCIER, C., FRANTZ, B. M., AND WHELAN, W. J. (1972) Eur. J. Bioehem. 26, l-9. 5. GUNJA-SMITH, Z., MARSHALL, J. J., SMITH, E. E., AND WHELAN, W. J. (1970) FEBS Lett. 12, 101-104. 6. RICHARDS, G. N., AND WHELAN, W. J. (1973) Carbohyd. Res. 27, 185-191. 7. MARSHALL, J. J., AND WHELAN, W. J. (1970) FEBS Lett. 9, 85-88. 8. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350-356. 9. HIZUKURI, S., TAKEDA, Y., AND IMAMURA, S. (1972) J. Agr. Chem. Sot. Japan 46, 119. 10. FRENCH, D. (1972) J. Japan. Starch Sci. 19, 8-25; KAINUMA, K., AND FRENCH, D. (1972) Biopolymers 11, 2241-2250. 11. KIKUMOTO, S., NIMURA, N., HIRAGA, Y., AND

12. 13. 14.

15. 16.

17.

18.

19. 20.

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KINOSHITA, T. (19’78) Carbohyd. Res. 61, 369-375. PEAT, S., WHELAN, W. J., AND THOMAS, G. J. (1952) J. Chem. Sot., 4546-4548. SUMMER, R., AND FRENCH, D. (1956) J. Biol. Chem. 222, 469-477. LEE, E. Y. C., MERCIER, C., AND WHELAN, W. J. (1968) Arch. Biochem. Biophys. 125, 1028- 1030. MERCIER, C., AND WHELAN, W. J. (1970) Eur. J. Biochem. 16, 579-583. LEE, E. Y. C., AND WHELAN, W. J. (1971) in The Enzymes (Boyer, P. D., ed.), Vol. 5, pp. 191-234, Academic Press, New York. GUNJA-SMITH, Z., MARSHALL, J. J., MERCIER, C., SMITH, E. E., AND WHELAN, W. J. (1970) FEBS. Lett. 12, 101-104. KRISMAN, C. R. (1962) Biochim. Biophys. Acta 65, 307; KJOLBERG, O., AND MANNERS, D. J. (1963) Biochem. J. 86, 1OP; DRUMMOND, G. S., SMITH, E. E., AND WHELAN, W. J. (1972) Eur. J. Biochem. 26, 168-176. Wu, H.-C. H., AND SARKO, A. (1978) Carbohyd. Res. 61, 7-25. Wu, H.-C. H., AND SARKO, A. (1978) Carbohyd. Res. 61, 27-40.

The mechanism of Q-enzyme action and its influence on the structure of amylopectin.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 198, No. 2, December, pp. 627-631, 1979 The Mechanism of Q-Enzyme Action and Its Influence Structure of...
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