- Email: [email protected]
The biosynthesis of starch
49
THE BIOSYNTHESIS PART XIII*.
INHIBITION
J. HOLLY,E. LAsz~6,
AND
OF STARCH
OF POTATO A.
PHOSPHORYLsASE
BY DONOR SUBSTRATE
ANALOGUES**
HOSCHKE
Institute of Agricultural Chemical Technology,
University of Technical Sciences, Budapest (Hungary)
(Received August 5th, 1968; in revised form, September 14th. 1968)
ABSTRACT
The inhibitory effect of D-glucose, 2-deoxy-D-arabino-hexose, 3-O-methyl-oglucose, and 6-O-methyl-D-glucose on the enzymic synthesis catalysed by potato phosphorylase has been investigated. Kinetic methods showed that, for both of the substrates employed, a competitive inhibitor effect was exerted by each of the substrate analogues examined. Values for Ki and Km calculated from the experimental data indicate that, in the presence of each of the added substrate analogues, the affinity of the enzyme for both substrates decreased. On the basis of the changes in Ki values, it appeared that, in the case of each substrate, the inhibitor effect of D-glucose was suppressed by the absence of the hydroxyl groups at the positions 2 and 3. INTRODU~ION
In earlier work on the mechanism of the action of potato phosphorylase, kinetic methods were used to determine the functional groups responsible for the binding of the substrate’v’. These groups were identified by inhibition iests2*‘. Later, in order to investigate the nature of the interacting groups in the substrate, various derivatives of o-glucose and D-glucopyranosyl phosphate were prepared4, and their effec;t on the enzyme reaction was studied’. This paper reports the inhibitory effects of donor substrate analogues. Previous papers in this field discuss only the effect of D-glucose, but the results are contradictory. According to Green and Stumpf6, Summer7, Ran8, and Nakamnra’, the enzymic reaction is not inhibited by D-glucose, whereas Fischer and Hilpert”, Arreguin”, and Porter” reported that D-glucose causes a competitive inhibition. Thus, prior to investigating the functional groups of the substrates, it appeared necessary to establish the nature of the inhibitory effect of D-glucose’. After these preliminary investigations, we examined the role of the hydroxyl groups at positions 2, 3, and 6, on the basis of the inhibition data obtained with suitable derivatives of D-glucose. . *Part XII: Stlrke, in the press. l*7lst Communication on polysaccharide research from this Department. Carbohyd. Res., 10 (1969) 49-56
J. HOLL6,
H)EXPERIMENTAL
AND
E. LhZL6,
A. HOSCHKE
RESULTS
Materials. Potato phosphoryiase was prefractionated with ammonium sulphate and purified by chromatography on DEAE-cellulose13 and had a specific activity of 450 E units per mg of protein. Amylopectin was an analytical grade (Calbiochem) product, and the D-glucopyranosyl phosphate (potassium salt, analytical grade) was a product of the Reanal Factory of Fine Chemicals, Budapest_ The following substrate analogues were used: D-glucose (analytical grade, Merck), 2-deoxyD-arabino-hexose (analytical grade, Reanal Factory of Fine Chemicals), 3-O-methyla-D-glUCOSe14,and 6-O-methyl-D-glucose (obtained from the Institute oi Organic Chemistry of L. Kossuth University, Debrecen). The substrates and the substrate analogues were employed in the inhibition experiments as 0.1~ solutions in a citrate buffer of pH 6.0. Determination of the initial velocity (vO). - The inhibition mixture, which contained the enzyme, the donor and acceptor substrates, and, in certain experiments, the corresponding substrate analogue, was placed, at the end of the reaction period, for 10 min in a water bath at 100”. After inactivation of the enzyme and removal of the denaturated protein by centrifugation, the amount of liberated inorganic phosphate was determined by the Fiske-Subbarow methodI as modified by Butenko and Kirsch”. From the quantity of liberated inorganic phosphate (first-order reaction) and the value of the equilibrium constant”, the initial velocity can be calculated. Determination of the mech.zmism of inhibition and of the inhibition constant. For the determination of the mechanism of inhibition, the graphical method suggested by Lineweaver 2nd Burk’ * was employed. Their equation for the inhibited reaction can be rearranged as follows: 1 -= Oi
$+m
K” VSI
[I
I,?1 f
7
where ui is the initial velocity of reaction, V, the maximum velocity of reaction, K, the apparent Michaelis constant, [a the substrate concentration, Ki the inhibition constant, and [a the inhibitor concentration. According to the above equation, the resulting straight lines intersect the ordinate at l/V,,,, and the abscissa at l/K,,,. The dissociation constant of the enzymeinhibitor complex, and the inhibition constant Ki can be calculated, in turn, from the slope of the graph. The linear equation for a non-inhibited system can be written in a completely analogous way. The values V,,, and K, can be determined in a manner analogous to the previous procedure_ Technique of inhibition experiments. - In this series of experiments, the enzymic reaction was inhibited by D-glucose and by D-glucose analogues substituted at the 2-, 3-, and 6-positions. Carbohyd.
Res.,
10 (1969)
49-56
In the course of the kinetic investigations, the concentrations of both D-ghcopyranosyl phosphate (as donor substrate) and of amylopectin (as. acceptor substrate) were varied, on applying two different, but constant, inhibitor concentrations. Initial velocities of reaction have been determined, in both cases, in noninhibited reaction systems (under identicaf conditions). The inhibiting effect of the substrate analogues was investigated only in the course of the enzymic synthesis catalysed by potato phosphorylase. It was established in the preliminary experiments that the phosphorolytic reaction is not inhibited by D-glucose and the above-mentioned substrate analogues’ g. ikestigation of the inhibition by D-g~zose. - In the synthesis reaction, the amount of inorganic phosphate liberated was determined at four different concentrations (3, 4, IO, and 20 mmoles per Iitre) of D-glucopyranosyl phosphate, at four different concentrations (0.095, 0.142, 0.237, and 0.950 mmole per We) of nonreducing end groups of amylopectin and at two D-glucose concentrations (0.3 and 0.6 mole per litre). The applied amount of enzyme was always 7.4 euzymic units, whilst the volume of reaction mixture totalled 2.0 mI. The foliowing digest conditions were employed: 0.1~ citrate buffer (pH 6.0) at 35 +0-l’ for 10 min. The vc values calculated from the measured data are listed in Table I. Values for l/c, in the inhibited and non-inhibited reaction mixtures plotted against donor and acceptor substrates, respectively, are presented in Fig. 1. TABLE
I
INHIBITING
EFFECT
OF
D-GLUCOSE
ON
INITIAL
VELOCITIES
AT
VARIOUS
SUBSTRATE
CONCENTRATIONS
Substrate concentration (mmoles/Iifre) zMIZucopyranosyi AmylopectirP phosphate
vo- 10-3 (pmolesjmin) With D-glucose Without any (0.6 mole) (0.3 mole) inhibitor
20 20 20 20 10 5 3
100.0 118.0 133.0 !93.0 164.0 156.0 130.0
aMmole
0.095 0.142 0.237 0.95 0.95 0.95 0.95
of
non-reducing
77.0 100.0 118.0 167.0 154.0 109.0 80.0
56.0 80.5 103.0 140.0 94.5 69.7 44.3
end-groups/litre.
Inuestigation of the inhibition by 2-deoxy-D-arabino-hexose. In this series of synthesis reactions, 0.3 and 0.6 mole of 2-deoxy-D-arabino-hexose were used as inhibitor. The v0 values calcnlated from the data of measurements are presented in Table 11. The reciprocal values of vc plotted against the reciprocal values of substrate concentrations are shown in Fig. 2. -Carbohyd. Res., 10 (1969) 49--56
3. HOLL6,
52
0.1
-OS
1
E. LkZL&,
ki. HOSCHKE
h
Fig. 1. Initial rate plotted against donor and acceptor substrate concentrations in the presence of n-glucose inhibitor. 1, No inhibitor; 2, with 0.3~ D-glucose; 3, with 0.6~ D-glucose; a, D-ghCOpyranosyl phosphate concentration varying from 3 to 20 mmoIes/Iitre at a constant amylopectin concentration of 0.95 mmole of non-reducing end-group/Iitre; b, amyIopectin concentration varying from 0.095 to 0.95 mmole of non-reducing end-group/litre at a constant D-ghCOpyrBIIOSyl phosphate concentration of 20 mmoles/Iitre. b
0
I ‘1%
-5
5
Fig. 2. Initial rate plotted against donor and acceptor substrate concentration in the presence of 2-deoxy-D-arabino-hexose inhibitor. I, No inhibitor; 2, with 0.3M 2-deoxy-D-orabino-hexese; 3, with 0.6~ 2-deoxy-D-arabino-hexose; a and b. see legend for Fig. I. TABLE INHIBITING
II EFFECT
OF
2-DEOXY-D-U~c2bino-HEXOSE
ON
INITIAL
VELOCITIES
AT
VARIOUS
SUBSTRATE
CONCENTRATIONS
Substrate
concentration
(mmolesj fitre)
D-Ghtcopyranosyl phosphate
AmylopectitP
20 20 20 20 10 5 3
0.095 0.142 0.237 0.95 0.95 0.95 0.95
ahfmole of non-reducing end-groupsflitre.
Curbohyci. Res., 10 (1969) 49-56
vo - IO-= @moiesfmin) With &Deoxy-D-arabinoWithout any inhibitor hexose (0.6 mole) (0.3 mole) 100.0 !09.0 146.5 181.0 166.0 156.0 138.0
95.5 109.0 136.0 176.5 162.0 141.5 116.5
73.2 88.5 123.0 148.0 146.0 113.0 95.0
STARCH
53
BIOSyEFIIIEsIs.xIrI
Invesiigation of the inhibition by 3-O-methyl-D-glucose. Because 6f the small amount of 3-O-methyl-D-glucose available, the inhibition was investigated only at one concentration (0.6~) of substrate analogue. The o0 values deterrined from the results obtained are given in Table III. Values of I/v0 plotted against the Go substrate concentrations are shown in Fig. 3. TABLE INHIBITING
III EFFECT
OF
%f%METHYL-D-GLUCOSE
ON
INITIAL
VELOCITIES
AT
VARIOUS
SUBSTRATE
CONCENTRATIONS
Substrate concentration (mmoles/litre) D-Ghicopyranosyl AmylopecGP phosphate
vo* 1O-3 (_umoiesfmin) Without any With 0.6 mole inhibitor of 3-0-methy& D-ghicose
105.0
0.095 0.142 0.237 0.95 0.95
20 20 20 20 10
5
0.95
3
0.95
88.5 73.0 92.5 132.0
117.0 125.0 153.0 138.0 136.0
115.0 118.0 77.5
102.0
=Mmole of non-reducing end-groups per litre. b
-0.5
Ql
‘h
-5
5
‘/[sl
Fig. 3. Initial rate plotted against donor and acceptor substrate concentration in the presence cf 3-O-methyl-D-glucose inhibkor. 1, No inhibitor; 2, with 0.6~ 3-O-methyl-D-glucose; a and b, see legend for Fig. 1.
Investigation of the inhibition by 6-O-methyl-D-glucose. - Again, the effect of inhibition has been investigated only at one concentration (0.3M) of substrate analogue. On varying the donor substrate concentration (20, 10, 5, and 3 mmoIes per litre), the acceptor substrate concentration was maintained constant (CL095 mmole per We). On varying the concentration of amylbpectin (0.095, 0.142, 0.237, and CWS mmole or” non-reducing end-groups per Ikej, the concentration of D-glucopyranosyl phosphate was kept constant (20 mmoles p& litre). Carbohyd. Res., 10 (1959) 49-56
-54
J. HOLti,
E.
LkSZL6,
li.
HGSCHRE
The calcuIated values of ~~ are presented in Table IV; and the values of l/v0 piotted against substrate concentrations are shown in Fig. 4. _b 0 +% 1
-5
Fig. 4.
lnitial
rate plotted
6-O-methyl-D-glucose
against
inhibitor.
5
donor and acceptor substrate concentration in the presence of inhibitor; 2, with 0.3~ 6-O-methyl-D-glucose: a and b, sfx
I, No
legend for Fig. 1. TABLE
IV
INHIBITING
EFFECT OF 6-O-METHYL-D-GLUCOSE
ON INITIAL
vELOCITIES AT VARIOUS SUBSTRATE
CONCENTRATIONS Substrate
vo .I O-3 @moleslmin)
concentration
D-Glttcopyranosyl phosphate
AmyiopectitP
Without any inhibitor
20 20 20 20 10 5 3
0.095 0.142 0.237 0.95 0.095 0.095 0.095
100.0 118.0 133.0 193.0 92.0 79.5 71.0
(mm0 fes/litrej
=Mmoie of non-reducing
end-groups
With 0.3 mole of &O-methylD-gkose
74.0 91.5 111.0 160.0 57.3 38.5 -
per litre.
DISCUSSION
Evaluation of the remits of imestigations of iddition. - As mentioned above, the results obtained were plotted by the double, reciprocal, graphic method suggested by Lineweaver and Burk (Figs, l-4), and the values K,, K,, and V, were estabIished from the slope tangents of the piotted straight lines and from the horizontal and vert&l axes (Tabie V). From these results, the conclusion is drawn that the inhibition process is competitive in respect of both the applied donor and the acceptor substrates for each of the substrate anaiogue& etiployed. This is proved also by the fact that the vah.~s for V, were identical throughout the experiments. Carbohyd. Res., 10 (1969) 49-56
’
Al 2 .’ 5 A
8 B a k9 0.0 0.3
_ aMmole and jtmole of non-reducing end-groups.
6.O-Methyl-D-&lucosc 1.65 9.10
3020
1067
0.0 0#3 0.6
0.0
3.0Methyl-D-jjlucose
D-Glucose
1.60 2.15 3.33
:Znoles per/lirre)
0.130
0.650
0.770 0,520
0.170 0.115
Kf
D-Gl~rcopyrotrosylphospl~ale
2-Deox~D-crrubina-o-ll~xose 0.0 0.3 0.6
concentratiorr (niole/litre)
Inltbitor
1064 4.53 10.00
applied
0.3 0.6
Irrltlbftor
O,lbi -,
0.157 -
0.200 -
0.204 -
SLY)
KlI (jwiole per
0.094 0.145
0.093 0.213
0.097 0.117 0.168
0.220 0.330 0.500
per he)
(mlok
0.52
1.57
-
1.30 0.79
0.540 0.405
Antylopecrina Ki Km
VALUES FOR K,,,, Ki, AND Vtn CALCULATED RY THE GRAPHIC METHOD OP L~NEWEAWR AND BURK’S FOR VARIOUS SUIMXATE
TABLE V
0.190 -
-
0.180
0.202 -
0.204 -,
ANALOGUES
J.
56
HOLL6,
E. LhZL6,
A. HOSCHKE
The apparent Michaelis constants increased with the rise of inhibitor conceu tration both for the donor substrate and for the acceptor substrate. This means that, in the presence of an inhibitor, the affinity of the enzyme decreases for both types of substrate applied. It appears from the data in ‘Table V that, in the cqe of both substrates, the inhibitor effect of o-glucose is supyressed to a great extent by the absence of the hydroxyl groups a.t C-2 and C-3, though, in the latter case, it is likeiy that the steric hindrance of the methoxyl group also plays a role. The absence of a hydroxyl group at C-6, in tum, caused no alterations in the inhibitor effect of D-glucose with any of the substrates examined. REFERENCES 1 2 3 4
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
J. J. J. J.
HOLLY). E. LAszL~, AND A. HOSCHKE, Stdrke, 18 (1966) 337. HOLLY, E. LAszr6, AND J. Jutisz, Stcirke, 19 (1967) 285. HOLLY, E. mzrb, A.HOSCHK?Z,AND P.VARGA, Carbohyd.Res., 8 (1968)416. HoLL~,E.LA~zL~,E.USKERT,ANDA.TUF&N,AC~~ Chim.Acrrd.Sci.Hung.,inpress. J. Ho~rd,E. L.&sz~6, AND A.T~ti~,in press. D. E. GREEN, AND P. K. STIJMPF,J. Biol. Chem., 142 (1942) 355. J.B. SI.MMER,T.C.CHU,AND A.T.BEYER, Arch_Biochem.,26 (195O)l. J. S. RAN AND K. V. GIRI,ArcJz. Biochem. Biophys., 38 (1958) 231. M. NAKAMIJRA, J. Agr. Chem. Sot. Japan., 26 (1952) 267. E. H. FISCHER AND H. M. HELPER, Experientiu, 9 (1953) 176. B. ARREGUIN-LOZANOAND J.BoNNER,PI~~~ PhysioL.24 (1949) 720. H. K. PORTER AND W. R. REES, PIant Physiol., 29 (1954) 514. J. HoLL~,E.L~~zL~,AND &HOSCHKE, StZirke, 16(1964) 243. H.B. WOOD,H.V.DIEHL,AND H. G.FLETcHER,JR.,J. Amer. Chem.Soc.,79 (1959) 1986. G. A.BTJTENKOAND N.N.KIRscH, Lab. Guz.,9(1940)555. C. H. FISKE AND I.S. SUBBAROW, J. Bioi. Chem., 66 (1925) 375. J. Horr&E.LAszr& AND ~.HOSCHICE,AC~U Chim.Acad_ Sci_Ifung_,50(1966)351. H. LINEWEAVER AND D. BURK, J. Amer. Chem. Sot., 56 (1934) 658. E. J~&zL& Dissertation,Cand. Sci.Chem., Budapest,1967.
Curboh_~d.Res., 10 (1969) 49-56
THE BIOSYNTHESIS PART XIII*.
INHIBITION
J. HOLLY,E. LAsz~6,
AND
OF STARCH
OF POTATO A.
PHOSPHORYLsASE
BY DONOR SUBSTRATE
ANALOGUES**
HOSCHKE
Institute of Agricultural Chemical Technology,
University of Technical Sciences, Budapest (Hungary)
(Received August 5th, 1968; in revised form, September 14th. 1968)
ABSTRACT
The inhibitory effect of D-glucose, 2-deoxy-D-arabino-hexose, 3-O-methyl-oglucose, and 6-O-methyl-D-glucose on the enzymic synthesis catalysed by potato phosphorylase has been investigated. Kinetic methods showed that, for both of the substrates employed, a competitive inhibitor effect was exerted by each of the substrate analogues examined. Values for Ki and Km calculated from the experimental data indicate that, in the presence of each of the added substrate analogues, the affinity of the enzyme for both substrates decreased. On the basis of the changes in Ki values, it appeared that, in the case of each substrate, the inhibitor effect of D-glucose was suppressed by the absence of the hydroxyl groups at the positions 2 and 3. INTRODU~ION
In earlier work on the mechanism of the action of potato phosphorylase, kinetic methods were used to determine the functional groups responsible for the binding of the substrate’v’. These groups were identified by inhibition iests2*‘. Later, in order to investigate the nature of the interacting groups in the substrate, various derivatives of o-glucose and D-glucopyranosyl phosphate were prepared4, and their effec;t on the enzyme reaction was studied’. This paper reports the inhibitory effects of donor substrate analogues. Previous papers in this field discuss only the effect of D-glucose, but the results are contradictory. According to Green and Stumpf6, Summer7, Ran8, and Nakamnra’, the enzymic reaction is not inhibited by D-glucose, whereas Fischer and Hilpert”, Arreguin”, and Porter” reported that D-glucose causes a competitive inhibition. Thus, prior to investigating the functional groups of the substrates, it appeared necessary to establish the nature of the inhibitory effect of D-glucose’. After these preliminary investigations, we examined the role of the hydroxyl groups at positions 2, 3, and 6, on the basis of the inhibition data obtained with suitable derivatives of D-glucose. . *Part XII: Stlrke, in the press. l*7lst Communication on polysaccharide research from this Department. Carbohyd. Res., 10 (1969) 49-56
J. HOLL6,
H)EXPERIMENTAL
AND
E. LhZL6,
A. HOSCHKE
RESULTS
Materials. Potato phosphoryiase was prefractionated with ammonium sulphate and purified by chromatography on DEAE-cellulose13 and had a specific activity of 450 E units per mg of protein. Amylopectin was an analytical grade (Calbiochem) product, and the D-glucopyranosyl phosphate (potassium salt, analytical grade) was a product of the Reanal Factory of Fine Chemicals, Budapest_ The following substrate analogues were used: D-glucose (analytical grade, Merck), 2-deoxyD-arabino-hexose (analytical grade, Reanal Factory of Fine Chemicals), 3-O-methyla-D-glUCOSe14,and 6-O-methyl-D-glucose (obtained from the Institute oi Organic Chemistry of L. Kossuth University, Debrecen). The substrates and the substrate analogues were employed in the inhibition experiments as 0.1~ solutions in a citrate buffer of pH 6.0. Determination of the initial velocity (vO). - The inhibition mixture, which contained the enzyme, the donor and acceptor substrates, and, in certain experiments, the corresponding substrate analogue, was placed, at the end of the reaction period, for 10 min in a water bath at 100”. After inactivation of the enzyme and removal of the denaturated protein by centrifugation, the amount of liberated inorganic phosphate was determined by the Fiske-Subbarow methodI as modified by Butenko and Kirsch”. From the quantity of liberated inorganic phosphate (first-order reaction) and the value of the equilibrium constant”, the initial velocity can be calculated. Determination of the mech.zmism of inhibition and of the inhibition constant. For the determination of the mechanism of inhibition, the graphical method suggested by Lineweaver 2nd Burk’ * was employed. Their equation for the inhibited reaction can be rearranged as follows: 1 -= Oi
$+m
K” VSI
[I
I,?1 f
7
where ui is the initial velocity of reaction, V, the maximum velocity of reaction, K, the apparent Michaelis constant, [a the substrate concentration, Ki the inhibition constant, and [a the inhibitor concentration. According to the above equation, the resulting straight lines intersect the ordinate at l/V,,,, and the abscissa at l/K,,,. The dissociation constant of the enzymeinhibitor complex, and the inhibition constant Ki can be calculated, in turn, from the slope of the graph. The linear equation for a non-inhibited system can be written in a completely analogous way. The values V,,, and K, can be determined in a manner analogous to the previous procedure_ Technique of inhibition experiments. - In this series of experiments, the enzymic reaction was inhibited by D-glucose and by D-glucose analogues substituted at the 2-, 3-, and 6-positions. Carbohyd.
Res.,
10 (1969)
49-56
In the course of the kinetic investigations, the concentrations of both D-ghcopyranosyl phosphate (as donor substrate) and of amylopectin (as. acceptor substrate) were varied, on applying two different, but constant, inhibitor concentrations. Initial velocities of reaction have been determined, in both cases, in noninhibited reaction systems (under identicaf conditions). The inhibiting effect of the substrate analogues was investigated only in the course of the enzymic synthesis catalysed by potato phosphorylase. It was established in the preliminary experiments that the phosphorolytic reaction is not inhibited by D-glucose and the above-mentioned substrate analogues’ g. ikestigation of the inhibition by D-g~zose. - In the synthesis reaction, the amount of inorganic phosphate liberated was determined at four different concentrations (3, 4, IO, and 20 mmoles per Iitre) of D-glucopyranosyl phosphate, at four different concentrations (0.095, 0.142, 0.237, and 0.950 mmole per We) of nonreducing end groups of amylopectin and at two D-glucose concentrations (0.3 and 0.6 mole per litre). The applied amount of enzyme was always 7.4 euzymic units, whilst the volume of reaction mixture totalled 2.0 mI. The foliowing digest conditions were employed: 0.1~ citrate buffer (pH 6.0) at 35 +0-l’ for 10 min. The vc values calculated from the measured data are listed in Table I. Values for l/c, in the inhibited and non-inhibited reaction mixtures plotted against donor and acceptor substrates, respectively, are presented in Fig. 1. TABLE
I
INHIBITING
EFFECT
OF
D-GLUCOSE
ON
INITIAL
VELOCITIES
AT
VARIOUS
SUBSTRATE
CONCENTRATIONS
Substrate concentration (mmoles/Iifre) zMIZucopyranosyi AmylopectirP phosphate
vo- 10-3 (pmolesjmin) With D-glucose Without any (0.6 mole) (0.3 mole) inhibitor
20 20 20 20 10 5 3
100.0 118.0 133.0 !93.0 164.0 156.0 130.0
aMmole
0.095 0.142 0.237 0.95 0.95 0.95 0.95
of
non-reducing
77.0 100.0 118.0 167.0 154.0 109.0 80.0
56.0 80.5 103.0 140.0 94.5 69.7 44.3
end-groups/litre.
Inuestigation of the inhibition by 2-deoxy-D-arabino-hexose. In this series of synthesis reactions, 0.3 and 0.6 mole of 2-deoxy-D-arabino-hexose were used as inhibitor. The v0 values calcnlated from the data of measurements are presented in Table 11. The reciprocal values of vc plotted against the reciprocal values of substrate concentrations are shown in Fig. 2. -Carbohyd. Res., 10 (1969) 49--56
3. HOLL6,
52
0.1
-OS
1
E. LkZL&,
ki. HOSCHKE
h
Fig. 1. Initial rate plotted against donor and acceptor substrate concentrations in the presence of n-glucose inhibitor. 1, No inhibitor; 2, with 0.3~ D-glucose; 3, with 0.6~ D-glucose; a, D-ghCOpyranosyl phosphate concentration varying from 3 to 20 mmoIes/Iitre at a constant amylopectin concentration of 0.95 mmole of non-reducing end-group/Iitre; b, amyIopectin concentration varying from 0.095 to 0.95 mmole of non-reducing end-group/litre at a constant D-ghCOpyrBIIOSyl phosphate concentration of 20 mmoles/Iitre. b
0
I ‘1%
-5
5
Fig. 2. Initial rate plotted against donor and acceptor substrate concentration in the presence of 2-deoxy-D-arabino-hexose inhibitor. I, No inhibitor; 2, with 0.3M 2-deoxy-D-orabino-hexese; 3, with 0.6~ 2-deoxy-D-arabino-hexose; a and b. see legend for Fig. I. TABLE INHIBITING
II EFFECT
OF
2-DEOXY-D-U~c2bino-HEXOSE
ON
INITIAL
VELOCITIES
AT
VARIOUS
SUBSTRATE
CONCENTRATIONS
Substrate
concentration
(mmolesj fitre)
D-Ghtcopyranosyl phosphate
AmylopectitP
20 20 20 20 10 5 3
0.095 0.142 0.237 0.95 0.95 0.95 0.95
ahfmole of non-reducing end-groupsflitre.
Curbohyci. Res., 10 (1969) 49-56
vo - IO-= @moiesfmin) With &Deoxy-D-arabinoWithout any inhibitor hexose (0.6 mole) (0.3 mole) 100.0 !09.0 146.5 181.0 166.0 156.0 138.0
95.5 109.0 136.0 176.5 162.0 141.5 116.5
73.2 88.5 123.0 148.0 146.0 113.0 95.0
STARCH
53
BIOSyEFIIIEsIs.xIrI
Invesiigation of the inhibition by 3-O-methyl-D-glucose. Because 6f the small amount of 3-O-methyl-D-glucose available, the inhibition was investigated only at one concentration (0.6~) of substrate analogue. The o0 values deterrined from the results obtained are given in Table III. Values of I/v0 plotted against the Go substrate concentrations are shown in Fig. 3. TABLE INHIBITING
III EFFECT
OF
%f%METHYL-D-GLUCOSE
ON
INITIAL
VELOCITIES
AT
VARIOUS
SUBSTRATE
CONCENTRATIONS
Substrate concentration (mmoles/litre) D-Ghicopyranosyl AmylopecGP phosphate
vo* 1O-3 (_umoiesfmin) Without any With 0.6 mole inhibitor of 3-0-methy& D-ghicose
105.0
0.095 0.142 0.237 0.95 0.95
20 20 20 20 10
5
0.95
3
0.95
88.5 73.0 92.5 132.0
117.0 125.0 153.0 138.0 136.0
115.0 118.0 77.5
102.0
=Mmole of non-reducing end-groups per litre. b
-0.5
Ql
‘h
-5
5
‘/[sl
Fig. 3. Initial rate plotted against donor and acceptor substrate concentration in the presence cf 3-O-methyl-D-glucose inhibkor. 1, No inhibitor; 2, with 0.6~ 3-O-methyl-D-glucose; a and b, see legend for Fig. 1.
Investigation of the inhibition by 6-O-methyl-D-glucose. - Again, the effect of inhibition has been investigated only at one concentration (0.3M) of substrate analogue. On varying the donor substrate concentration (20, 10, 5, and 3 mmoIes per litre), the acceptor substrate concentration was maintained constant (CL095 mmole per We). On varying the concentration of amylbpectin (0.095, 0.142, 0.237, and CWS mmole or” non-reducing end-groups per Ikej, the concentration of D-glucopyranosyl phosphate was kept constant (20 mmoles p& litre). Carbohyd. Res., 10 (1959) 49-56
-54
J. HOLti,
E.
LkSZL6,
li.
HGSCHRE
The calcuIated values of ~~ are presented in Table IV; and the values of l/v0 piotted against substrate concentrations are shown in Fig. 4. _b 0 +% 1
-5
Fig. 4.
lnitial
rate plotted
6-O-methyl-D-glucose
against
inhibitor.
5
donor and acceptor substrate concentration in the presence of inhibitor; 2, with 0.3~ 6-O-methyl-D-glucose: a and b, sfx
I, No
legend for Fig. 1. TABLE
IV
INHIBITING
EFFECT OF 6-O-METHYL-D-GLUCOSE
ON INITIAL
vELOCITIES AT VARIOUS SUBSTRATE
CONCENTRATIONS Substrate
vo .I O-3 @moleslmin)
concentration
D-Glttcopyranosyl phosphate
AmyiopectitP
Without any inhibitor
20 20 20 20 10 5 3
0.095 0.142 0.237 0.95 0.095 0.095 0.095
100.0 118.0 133.0 193.0 92.0 79.5 71.0
(mm0 fes/litrej
=Mmoie of non-reducing
end-groups
With 0.3 mole of &O-methylD-gkose
74.0 91.5 111.0 160.0 57.3 38.5 -
per litre.
DISCUSSION
Evaluation of the remits of imestigations of iddition. - As mentioned above, the results obtained were plotted by the double, reciprocal, graphic method suggested by Lineweaver and Burk (Figs, l-4), and the values K,, K,, and V, were estabIished from the slope tangents of the piotted straight lines and from the horizontal and vert&l axes (Tabie V). From these results, the conclusion is drawn that the inhibition process is competitive in respect of both the applied donor and the acceptor substrates for each of the substrate anaiogue& etiployed. This is proved also by the fact that the vah.~s for V, were identical throughout the experiments. Carbohyd. Res., 10 (1969) 49-56
’
Al 2 .’ 5 A
8 B a k9 0.0 0.3
_ aMmole and jtmole of non-reducing end-groups.
6.O-Methyl-D-&lucosc 1.65 9.10
3020
1067
0.0 0#3 0.6
0.0
3.0Methyl-D-jjlucose
D-Glucose
1.60 2.15 3.33
:Znoles per/lirre)
0.130
0.650
0.770 0,520
0.170 0.115
Kf
D-Gl~rcopyrotrosylphospl~ale
2-Deox~D-crrubina-o-ll~xose 0.0 0.3 0.6
concentratiorr (niole/litre)
Inltbitor
1064 4.53 10.00
applied
0.3 0.6
Irrltlbftor
O,lbi -,
0.157 -
0.200 -
0.204 -
SLY)
KlI (jwiole per
0.094 0.145
0.093 0.213
0.097 0.117 0.168
0.220 0.330 0.500
per he)
(mlok
0.52
1.57
-
1.30 0.79
0.540 0.405
Antylopecrina Ki Km
VALUES FOR K,,,, Ki, AND Vtn CALCULATED RY THE GRAPHIC METHOD OP L~NEWEAWR AND BURK’S FOR VARIOUS SUIMXATE
TABLE V
0.190 -
-
0.180
0.202 -
0.204 -,
ANALOGUES
J.
56
HOLL6,
E. LhZL6,
A. HOSCHKE
The apparent Michaelis constants increased with the rise of inhibitor conceu tration both for the donor substrate and for the acceptor substrate. This means that, in the presence of an inhibitor, the affinity of the enzyme decreases for both types of substrate applied. It appears from the data in ‘Table V that, in the cqe of both substrates, the inhibitor effect of o-glucose is supyressed to a great extent by the absence of the hydroxyl groups a.t C-2 and C-3, though, in the latter case, it is likeiy that the steric hindrance of the methoxyl group also plays a role. The absence of a hydroxyl group at C-6, in tum, caused no alterations in the inhibitor effect of D-glucose with any of the substrates examined. REFERENCES 1 2 3 4
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
J. J. J. J.
HOLLY). E. LAszL~, AND A. HOSCHKE, Stdrke, 18 (1966) 337. HOLLY, E. LAszr6, AND J. Jutisz, Stcirke, 19 (1967) 285. HOLLY, E. mzrb, A.HOSCHK?Z,AND P.VARGA, Carbohyd.Res., 8 (1968)416. HoLL~,E.LA~zL~,E.USKERT,ANDA.TUF&N,AC~~ Chim.Acrrd.Sci.Hung.,inpress. J. Ho~rd,E. L.&sz~6, AND A.T~ti~,in press. D. E. GREEN, AND P. K. STIJMPF,J. Biol. Chem., 142 (1942) 355. J.B. SI.MMER,T.C.CHU,AND A.T.BEYER, Arch_Biochem.,26 (195O)l. J. S. RAN AND K. V. GIRI,ArcJz. Biochem. Biophys., 38 (1958) 231. M. NAKAMIJRA, J. Agr. Chem. Sot. Japan., 26 (1952) 267. E. H. FISCHER AND H. M. HELPER, Experientiu, 9 (1953) 176. B. ARREGUIN-LOZANOAND J.BoNNER,PI~~~ PhysioL.24 (1949) 720. H. K. PORTER AND W. R. REES, PIant Physiol., 29 (1954) 514. J. HoLL~,E.L~~zL~,AND &HOSCHKE, StZirke, 16(1964) 243. H.B. WOOD,H.V.DIEHL,AND H. G.FLETcHER,JR.,J. Amer. Chem.Soc.,79 (1959) 1986. G. A.BTJTENKOAND N.N.KIRscH, Lab. Guz.,9(1940)555. C. H. FISKE AND I.S. SUBBAROW, J. Bioi. Chem., 66 (1925) 375. J. Horr&E.LAszr& AND ~.HOSCHICE,AC~U Chim.Acad_ Sci_Ifung_,50(1966)351. H. LINEWEAVER AND D. BURK, J. Amer. Chem. Sot., 56 (1934) 658. E. J~&zL& Dissertation,Cand. Sci.Chem., Budapest,1967.
Curboh_~d.Res., 10 (1969) 49-56