The action of β-amylase on some unusual oligosaccharides

CARBOHYDRATE

The action of /I-amylase

on some

unusual

RESEARCH

oligosaccharides

&Amy&e catalyzes the stepwise hydrolyses of maltose from the nonreducmg end of a malto-ohgosacchande The nature of its action on natrve-starch fractrons and on ohgosacchandes has been summamd by FrenchI. More recently, Thoma and Koshlandz have reported on extensive studres of its actrons on methyl a-maltoohgosaccharides Cbenncally motied amyloses have also been used as substrates for /3-amylasej 4 The action of the enzyme on such polymers, In whzch one or more of the D-glucose residues has heen modrfied, could yield mformatron on the nature of those substrate groups that are necessary m the bmdmg or hydrolysis steps The present mvestrgatzon is concerned wzth the action of cryscaihne, sweet-potato /?-amylase on some low molecular-werght ohgosacchandes contarmng one pyranoszde moretywhrch drffers slightly from a o-glucopyranoside residue These molecules are shown m Frgure I The trrsaccharrde lb lacks the hydroxymethyl group on C-5 of the methyl a-D-PyranOsrde resrdue Tetrasaccharide 2b lacks the same group The tetrasaccharrde 2c has a methyl a-deoxy-a-D-arabizzo-hexopyranosrde residue A pentasaccharrde, methyl O-a-maltotetraosyl-(1~4)-2-deoxy-a-D-ar~~-hexopyranonde, was also used as a substrate for the crystalline enzyme RESULTS AND

DISCUSSION

The action of crystallme, sweet-potato /3-amylase on small samples of 2b and 2c was observed by examimng thin-layer chromatograms of the reactron mrxtures Both of these products were completely hydrolyzed to maltose and the respectrve methyl O-a-D-glucopyranosyl-( r -+4)-a--D-aldopyranosrde The pentasaccharrde methyl O-a-D-maltotetraosyl-(1 +$a-deoxy-&-D-az&abmo-hexopyranosld liberated maltose and the tnsaccharzde lc When the concentration of enzyme was increased by a factor of one hundred, the Ic was further cleaved, to maltose and methyl a-deoxya-D-arabzzzo-hexopyranosrde That thrs great increase In concentratron of enzyme was necessary m order to accomphsh this latter hydrolysis is not consrdered to be parucularly important, Peat, Whelan, and Jones ’ have prevrously demonstrated that low concentratrons of /3-amylase do not liberate maltose from either maltotnose or unmochfied la The tnsacchande (lb) contammg the D-xylopyranosrde resrdue was completely resrstant to the actron of the enzyme, even when I g mg of enzyme was used per I o mg of substrate Saccharides 2b and 2c were compared wrth unmoddled 2a as substrates for @unyIase by measurmg the hberatron of maltose Figure 2 shows that the unmodrfied 2a 1s the most readily hydrolyzed substrate, but rt also mdrcates that the enzyme IS reasonably active toward 2c It would seem that the absence of the hydroxyl group at C-2 of the methyl a-aldopyranosrde residue does not prevent thrs momed methyl Carbohydrate

Res ,3 (1966) 2tp-245

243

NOTES

maltotetraoslde from servmg as a substrate These data, and the previously demonstrated hydrolyses of lc, show that the presence of a hydroxyl group at C-Z IS not essential for enzyme actlon as long as there ISa termmal maltose residue that can be hberated.

OH

OH

X

(2)

R = CHzOH, X = OH, lb, methyl a-maltosyl-(t-+&c-or-Dmethyl a-makosyl-(x+&2-deoxy-z-D-“gbopyranoslde”, R = CHzOH, X = H, 2a, methyl a-maltotetraoslde, R = CHzOH, X = OH, Zb, methyl a-maltotnosyl(r-t&a-D-xylopyranoslde, R = H, X = OH, and Zc, methyl a-maltotriosyl-(r-+&2-deoxy-c+nX=H “glucopyranoslde”, R=CHzOH, Fxg

I

la,

Methyl

cc-maltornoslde,

xylopyranoslde, R = H, X = OH, lc,

Figure 2 also shows that the p-amylase has very httle actlvlty on 2b at the speafied concentration of enzyme. When the amylase concentration was mcreased by a factor of twenty, there was 60% hydrolysis in two hours These data, and the prevrously demonstrated stab&y of lb m the presence of very hgh concentrations of Bamylase, suggest that the presence of the hydroxymethyl group at C-5 of the pyranoslde residue ISmuch more Important to the actton of the enzyme than ISthe hydroxyl group at C-2 The reststance of lb to hycirolysts mrght have been caused by its farlure to be bound by the /I-amylase However, Frgure 3 shows that lb serves as a competltlve mlubitor of the actlon of the crystalhne, sweet-potato amylase on corn amylose Inhrbmons of thus sort are usually Interpreted as demonstratmg reversrble bmdmg The drfTerence m behavior between the two driTerentstructural modrficatlons permits the speculatron that thus may be an rllustration of Kosbland’? “mduced fit”. It seems possrble that the absence of the hydroxymethyl group at C-5 causes a more serrous mrsahgnment of the acttve center of the enzyme than does the absence of the hydroxyl group at C-2 The Importance of the hydroxymethyl group has been further shown by the farlure of fl-amylase to catalyze the hydrolysis of an amylose molecule that we have prepared m which some of the primary hydroxyl groups are methylated Whether thts xmsahgnment IS a result of sample stenc factors or of hydrogen bondmg may be at least partially clanfied by results obtamed wrth some other modrfieatrons now in preparation Carbohydrate Res , 3 (1966) 242-245

244

100

1

20 . 0

15

30

60 Mmutes

120

F,g 2 Actlon of crystalhne, sweet-potato amjlase on Za, Zb, and 2c A 2a, l Zb,Q 2c Percent of hydrolysis IS calculated as the percent of the theoretlcal yield of maltose Enzyme concentration 1s I 5 x IO-4 mg per mg of substrate Fig 3 Compound lb as an mhlbltor of the actlon of crystalhne, sweet-potato amylase on amylose, a Lmeweaver-Burke plotlo . p-amylase on amylose o /3-amylase on amyiose, with a concentratlonof lb of I o mg/ml EXPERIMENTAL

The corn amylose was a gtft of the A E Staley Co (Decatur, Ilhnois) Crystaline, sweet-potato &amylase was obtamed from Worthmgton Brochemrcal Cot-p (Freehold, New Jersey) Methyl a-maltotnosrde and a-tetraosrde were prepared and fractronated by the techmque of Pazur, Marsh, and Ando7. The molfied methyl cr-ohgosacchandes were prepared by the techmque of Wheeler, Hanke, and Weill’, and were fractronated by the techmque of Pazur et aZ7. In each case, the isolated fractron was shown to be homogeneous (t 1 c ) by comparison with the senes prepared by the method of Wheeler et al8

Methyl O-a-maltotnosyl-(I -&a-D-xylopyranoslde, 2b, was obtained m a crystalline and an amorphous form Samples of these and of the methyl O-a-maltosyl(r-+4)-a-D-xylopyranoside, lb, were sent to Weller and Strauss (Oxford, England) for analysis aH,O C, 42 o; H, 6.7. Found Anal. (2b, crystalline) Calc for C,,H,,O,, C, 42 I ; H, 6 g (2b, amorphous) Calc for C24H42020 3H20 C, 41 o; H, 6 8 Found C, 41 o; H, 6 8 (lb) Cak for C18H32015-2HZ0. C, 41 3, H, 6 g Found C, 414, H, 7 3 T 1 c expenments were conducted on slhca gel G (Warner Ch~lcott Laboratory, Instrument Divmon, Morrts Plams, New Jersey) The solvent imganon system was ethanol-ether-toluene (40 50 IO, v/v) The colors were developed by spraying wrth a 5 % solution of concentrated sulfuric acid m ethanol, followed by heatmg 111an oven at 105” for 30 min The t IX experiments wnh lb, 2b, 2c, and the pentasaccharrde were conducted Carbohydrate

Res , 3 (1966) w-%+5

NOTES

245

atpH 4 8 at 26O,with a substrate concentratron of I o mg per ml The enzyme concentratlon was 3 0 x 10~~ mg per mg of substrate for the expenments with 2b,2c, and the pentasacchande contarmng the a-deoxy-a-D-arabma-hexopyranos:de reszdue This concentratzon was Increased to 3 ox IO-’ mg per mg for the experiments with lc, and to 1.9 mg per mg for the expenments with lb.At the end of 48 h, the samples were heated m a bozlrng-water bath, and de-zomzed by passage through a column (I I xo g cm) of Amberhte MB-3 resm (mzxed amon- and catron-exchangers; Rohm and Haas Co , Phlladelphra, Pennsylvama) The respective effluents were concentrated to a small volume before apphcatron to the thm-layer plates The data for Flgure 2 were obtamed with a solution contalmng o 20 mg per ml of substrate m a o or6 M acetate buffer of pH 4 8. The /3-amylase concentratron was mg per mg of substrate Reducmg values were determmed by the method I gx 10-4 of Hagedorn and Jenseng, usmg appropnate converszon factors for maltose Vacuum&red amylose was dzssolved m a measured, small amount of 2M KOH, and the solutron was diluted, and neutrahzed wzth acetIc acid to pH 4 8. Enough acetate buffer and water were added so that the buffer concentration was 0.016~ at pH 4 8, the amylose concentratron was 2 mg per ml Enzyme activity was measured on four concentratrons of thus amylose m a solution which was o 016 M wnh respect to acetate buffer of pH 4 8. The enzyme concentratzon was I 2x IO-~ mg per ml Inhlbltron studzes with lb were conducted on solutzons contammg I o mg of lb per ml, m addrtron to the same amylose, buffer, and enzyme as were used m the absence of the m2ubitor ACKNOWLEDGMENT

Thrs work was supported by Grant GM 08927 of the Research Grants Branch of the National Instrtutes of General Medlcal Science, U S Pubhc Health Servzce C

Department of Chemrstry, Rutgers-The State Umversuy, Newark, New Jersey 07102 (U S A )

EDWIN

WEILL

ROBERT REBHAHN

REFERENCES I D FRENCH, Enzymes, 4 (1960) 345 2 J A THOMA AND E KOSHLAND, JR , J Bd 3 4 5 6 7 8 g IO

Chem

,235 (x960) 251 I, J Am Chem Sot ,82 (1960)

3329 E HUSEMANN AND E LINDEMANN, Staerke, 6 (1954) 141 B J BINES AND W J- WHELAN, C’henr Ind (London), (1960) gg7 S PEAT, W J WHELAN, AND G JONES, J Chem SOC, (x957) 2490 D. E KOSHLAND, JR, Enzymes, I (1960) 305 J PAZUR, J M MARSH,ANDT ANDO, J Am Chem Sot 81(1959)21’lo_ M WHEELER, P HANKE. AND C E WEILL, Arch Bmchem Brophys, 102 (1963) 397 H C HAGEDORN AND B N JENSON,~~~ PolarunefrySaccharrmetryadrhe Sugars, Std Clrc. C440, (1942) I98 H L INEWEAVER AND D BURK, J Am Chem Sot , 56 (1934) 658

Nat1

BU

(Recewed July 22nd. 1966) Carbohydrate Res , 3 (1966) 242-245