Planta (1990)182:546-552
PI~LI"]_t~ 9 Springer-Verlag1990
Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize* Stanton B. Dotson**, David A. Frisch, David A. Somers***, and Burle G. Gengenbach Department of Agronomyand Plant Genetics,and Plant MolecularGenetics Institute, University of Minnesota, St. Paul, MN 55108, USA Received 10 May; accepted 7 June 1990
Abstract. Aspartate kinase (AK; EC 2.7.2.4) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations, AskLT19 and Ask2-LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived amino acids in maize. Kernels of the homozygous Ask2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine, compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 ~tM L-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygous Ask and homozygous Ask2 mutants required 25 and 760 gM lysine for 50% inhibition, respectively, indicating that Ask and Ask2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the homozygous mutant Ask2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutant Ask2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor of Ask2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive inhibition of wildtype AK. Reduced lysine sensitivity of the Ask2 gene product appeared to * Scientific paper No. 17419, Minnesota Agricultural Experiment Station projects No. 0302-4813-56 and No. 0302-4818-32 ** Present address: Monsanto Company, 700 ChesterfieldVillage Parkway, Chesterfield,MO 63198, USA *** To whom correspondence should be addressed Abbreviations: AK(s)= aspartate kinase(s); LT = equimolar lysine plus threonine
reduce the lysine inhibition of all of the AK activity detected in homozygous Ask2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from the Ask and Ask2 structural genes.
Key words: Amino acid biosynthesis Aspartate kinase - Lysine - Mutant (threonine overproduction) - Threonine - Zea (amino acids)
Introduction As the first enzyme in the biosynthesis pathway for the aspartate-family amino acids, aspartate kinase (AK; EC 2.7.2.4) potentially exerts a key regulatory role in the synthesis of lysine, threonine, methionine and other derivatives of the pathway (Bryan 1980). Aspartate kinase isoforms in plants are feedback-inhibited by lysine or threonine, and S-adenosyl methionine can increase the inhibition caused by low concentrations of lysine (Bryan 1980; Rognes et al. 1980). Growth of cell cultures and seedlings of many plants is inhibited by lysine plus threonine (LT), presumably because of feedback inhibition of AK, and possibly other steps in the pathway causing growth-limiting methionine biosynthesis levels (Dunham and Bryan 1969; Green and Phillips 1974; Gengenbach 1984). Selection for resistance to toxic LT levels, therefore, was proposed as a possible means to obtain mutations of AK that would be less sensitive to feedback regulation (Green and Phillips 1974). Mutants resistant to LT have been selected in maize (Zea mays L.) (Hibberd et al. 1980; Hibberd and Green 1982; Miao et al. 1988; Diedrick et al. 1990), carrot (Daucus carota L.) (Cattoir-Reynaerts et al. 1983), barley (Hordeum vulgare L.) (Bright et al. 1982) and tobacco (Nicotiana tabacum L.) (Bourgin et al. 1982). Increased accumulation of threonine in the free-amino-acid pool often is associated with LT resistance. In barley, LT-resistant
S.B. Dotson et al. : Lysine-insensitive aspartate kinase in maize mutants m u t a n t s were s h o w n to result f r o m t w o n o n a l l e l i c genes e n c o d i n g m u t a n t f o r m s o f A K i s o e n z y m e s t h a t were less sensitive to lysine f e e d b a c k i n h i b i t i o n ( A r r u d a et al. 1984; B r i g h t et al. 1982; R o g n e s et al. 1983). Several L T - r e s i s t a n t m a i z e lines h a v e been selected f r o m tissue c u l t u r e ( H i b b e r d et al. 1980; H i b b e r d a n d G r e e n 1982; M i a o et al. 1988; D i e d r i c k et al. 1990). T h e initially selected L T - r e s i s t a n t cell line h a d A K activity in c r u d e e x t r a c t s t h a t was a b o u t e i g h t f o l d less sensitive to lysine i n h i b i t i o n t h a n A K f r o m u n s e l e c t e d L T - s u s c e p tible c u l t u r e s ( H i b b e r d et al. 1980). P l a n t s r e g e n e r a t e d f r o m this c u l t u r e were infertile so p r o g e n y were n o t o b t a i n e d for genetic tests a n d the r e l a t i o n s h i p b e t w e e n alt e r e d A K a n d t h r e o n i n e o v e r p r o d u c t i o n c o u l d n o t be f u r t h e r investigated. S u b s e q u e n t l y , a d d i t i o n a l m a i z e m u t a n t s were selected for L T resistance a n d s h o w e d simple, d o m i n a n t , M e n d e l i a n i n h e r i t a n c e either for high freet h r e o n i n e level ( F r i s c h a n d G e n g e n b a c h 1986; D i e d r i c k et al. 1990) o r for g r o w t h o f seedlings ( H i b b e r d a n d G r e e n 1982) o r excised r o o t tips in the presence o f L T ( M i a o et al. 1988). F r e e - t h r e o n i n e c o n c e n t r a t i o n s in the m u t a n t k e r n e l s r a n g e f r o m 10-fold to m o r e t h a n 100-fold h i g h e r t h a n c o r r e s p o n d i n g w i l d t y p e kernels d e p e n d i n g o n the i n b r e d b a c k g r o u n d a n d allele c o m p o s i t i o n in the e m b r y o a n d e n d o s p e r m ( H i b b e r d a n d G r e e n 1982; M i a o e t a l . 1988). Recently, t w o t h r e o n i n e - o v e r p r o d u c i n g m a i z e p h e n o t y p e s were s h o w n to be c o n t r o l l e d b y n o n a l lelic loci, Ask a n d Ask21 ( F r i s c h a n d G e n g e n b a c h 1986; D i e d r i c k et al. 1990). T h e s e m u t a n t s o v e r p r o d u c e a s p a r t a t e - d e r i v e d a m i n o a c i d s ; h o w e v e r , the b i o c h e m i c a l basis for these m a i z e m u t a n t s h a s n o t yet been c h a r a c t e r i z e d . T h e objective o f this s t u d y was to c h a r a c t e r i z e the A K a c t i v i t y f r o m the t w o L T - r e s i s t a n t m u t a n t m a i z e g e n o t y p e s , in o r d e r to u n d e r s t a n d the role o f this e n z y m e in r e g u l a t i n g the synthesis o f a s p a r t a t e - d e r i v e d a m i n o acids. L y s i n e - i n s e n s i t i v e f o r m s o f A K c o s e g r e g a t e d w i t h a m i n o - a c i d o v e r p r o d u c t i o n in b o t h m a i z e m u t a n t s , est a b l i s h i n g t h a t o v e r p r o d u c t i o n was c a u s e d b y m u t a t i o n s in the t w o A K s t r u c t u r a l loci. W e were able to derive a line h o m o z y g o u s for the Ask2 m u t a t i o n . P u r i f i e d Ask2 A K w a s h o m o g e n e o u s l y lysine-insensitive i n d i c a t i n g t h a t the g e n e - e n z y m e r e l a t i o n s h i p s o f m a i z e A K likely differed f r o m the discrete i s o e n z y m e c o m p o s i t i o n des c r i b e d for A K in b a r l e y ( A r r u d a et al. 1984; B r i g h t et al. 1982; R o g n e s et al. 1983). F u r t h e r m o r e , f e e d b a c k insensitive f o r m s o f A K c o n f e r r e d s u b s t a n t i a l o v e r p r o d u c t i o n o f t h r e o n i n e , lysine, m e t h i o n i n e a n d isoleucine, i n d i c a t i n g e l e v a t e d flux t h r o u g h the p a t h w a y a n d d e m o n s t r a t i n g t h a t A K p l a y s a k e y r e g u l a t o r y role for the p a t h w a y in maize.
Material and methods Mutant and wildtype genotypes. Maize (Zea mays L.) inbred line A619 served as the wildtype control and was used as the recurrent Locus and allele designations conform to guidelines for maize genetic nomenclature (Burnham et al. 1975) in which the first locus of a series is given a three-letter designation indicative of the phenotype (Ask for aspartate kinase) and succeeding loci are designated by the same letters plus a number (Ask2)
547 parent in developing the derivative mutant genotypes following selection for LT resistance in tissue culture. The mutant allele (AskLT19) for one AK (Ask) locus was derived from LT-resistant LT19 plants produced as described by Hibberd and Green (1982). Plants containing the Ask-LT19 allele (provisionally designated Ltr*19 by Hibberd and Green 1982) were backcrossed seven generations to wildtype A619 plants and then progeny were self-pollinated two generations to produce a limited amount of A619Ask-LT19/AskLT19 seed. Because few homozygous Ask seed were available, a uniform population of heterozygous A619Ask-LT19/+ plants was obtained by a final cross to A619 for the purpose of enzyme analysis. The mutant allele (Ask2-LT20) of the second AK locus (Ask2) was obtained from plants regenerated from another tissue culture selected for LT resistance (Diedrick et al. 1990). A homozygous mutant line of A619Ask2-LT20/Ask2-LT20 was obtained after backcrossing five generations to A6194./4-;+/4- followed by three generations of selfing. To simplify genotype designations throughout the remainder of this report, the genotypes with respect to the Ask and Ask2 loci are as follows: wildtype=A619 4-/4-; 4-/4- ; heterozygous Ask=A619Ask-LT19/ 4- ; 4-/4- ; and homozygous Ask2=A6194-/4-;Ask2-LT20/Ask2-LT20. These mutant genotypes were confirmed by analyses of test crosses to wildtype A619 in 1988. Seed supplies of both mutant lines were not adequate for enzyme extraction from seedlings; therefore, plants were grown to anthesis in the field in 1988 and enzyme extracted from immature ears. Also, since the experimental hypothesis was that increased kernel threonine was the result of an altered AK, it made sense to extract AK from a related tissue such as immature ears. Unpollinated, immature ears (3-8 cm) were removed from the husks and silks, quickly frozen and ground in liquid nitrogen using a stainless steel Waring blender, and stored under nitrogen at - 7 0 ~ C until enzyme purification.
Enzyme purification. Aspartate kinase from immature ears was extracted and purified as described for maize suspension-cell cultures (Dotson et al. 1989). Crude extracts were desalted, and partially purified AK was obtained by successive chromatographic steps on phenyl sepharose hydrophobic, Sephacryl S300HR gel filtration, and FPLC Mono Q anion-exchange columns. The buffers used for anion exchange were prepared in a single batch and used during the purification of AK from each genotype to allow comparison of elution profiles. Several purified preparations of AK from each source were combined (approx. 600 g FW total) to obtain enough enzyme for kinetic analyses. After ion-exchange chromatography, 2 mg/ml bovine serum albumin was added to each preparation to stabilize enzyme activity, and stored at - 7 0 ~ C. Aspartate-kinase assays. A modified aspartate-hydroxamate method was used during enzyme purification and the pyruvate kinaselactate dehydrogenase coupled assay was used for kinetic analysis (Dotson et al. 1989, 1990). Amino-acid analysis. Free and total concentrations of amino acids in kernels harvested from wildtype and homozygous Ask2 plants were extracted and analyzed as previously described (Diedrick et al. 1990). Data analysis. Aspartate-dependent AK activity was expressed in pkat (pmol-s -1) and analyzed according to graphical methods (Cleland 1967, 1970). Computer programs Eqordo and Hypero (Cleland 1979) were supplied by Charles B. Grissom (Department of Chemistry, University of California, Berkeley, USA) and used to fit substrate kinetic data and hyperbolic velocity data for lysine inhibition, respectively. The programs Uncomp, Comp and Noncomp were used as additional evidence to discriminate an uncompetitive versus a non-competitive inhibition and to derive the inhibition constants in Table 2. Lysine inhibition data also were fitted to the logarithmic Hill equation (Segal 1975) by plotting log[V/ (Vm.x-V)] versus log [lysine].
548
S.B. Dotson et al. : Lysine-insensitive aspartate kinase in maize mutants
Results
,.-, 3.0
Amino-acid composition. The free and total amounts of aspartate-derived amino acids in wildtype and homozygous m u t a n t Ask2 kernels are shown in Table 1. Free threonine, methionine, lysine, and isoleucine were increased 174-, 10-, 13-, and 2-fold, in the order given in the m u t a n t kernels c o m p a r e d to wildtype. The increased free threonine contributed to a significant increase in total kernel threonine. Increases in total methionine and lysine were associated with, but not fully attributable to the free-pool increases, indicating proteinb o u n d methionine or lysine composition also changed. No significant differences in total kernel composition were observed in other amino acids except for a 10-fold increase in free serine (data not shown). Heterozygous Ask kernels were previously shown to have a 29-fold increase in free threonine c o m p a r e d to wildtype (Hibberd and Green 1982). Enzyme purification. Aspartate kinase was partially purified f r o m immature ears o f wildtype, heterozygous Ask and homozygous Ask2 plants using a procedure that was developed for A K purification from Black Mexican Sweet maize suspension-culture cells and routinely resulted in 1200-fold purification with greater than 85% recovery (Dotson et al. 1989). Specific activities in desalted crude extracts from the immature ears of wildtype and the mutants were approx. 14 p k t a l . ( m g protein)-1 indicating that the increased threonine in the kernel was not the result of A K overexpression. Aspartate-kinase activity f r o m the three genotypes eluted from a Sephacryl S300HR gel-filtration column as single peaks of Mr about 254000 indicating nearly identical sizes. The A K activity from wildtype and homozygous Ask2 eluted f r o m the M o n o Q anion-exchange column as single peaks at similar positions in the elution gradient (Fig. 1). Aspartate kinase f r o m heterozygous Ask plants eluted as an early b r o a d shoulder followed by a major activity peak, presumably indicating a mixture of wildtype and
_~_~c2 . 0 v
._> 1.o < 0.0
Anion-exchange chromatography elution of AK activity from immature ears of wildtype A619, heterozygous Ask, and homozygous Ask2 maize. A Fast Protein Liquid Chromatography Mono Q column was eluted with a linear 75-275 mM KC1 gradient. o, Wildtype A619; o, Ask/4-; x , Ask2/Ask2 Fig. l.
mutant A K isoforms. Characterization of the major peak of A K purified from the Ask plants is reported herein.
Lysine inhibition of AK activities &olated from Ask and Ask2. Approximately 10 ~tM lysine was required to inhibit 50% of the A K activity 05o) from wildtype maize as determined from fit to the Hill equation (Fig. 2). In contrast, the I5o values for the A K activity from heterozygous Ask and homozygous Ask2 plants were 25 laM and 760 laM lysine, respectively. These results indicated that the amino-acid overproduction of the mutations is conferred by a reduction in lysine inhibition o f AK. Therefore, Ask and Ask2 appear to be independent loci encoding lysine-regulated subunits of AK. Hill coefficients were calculated for the wildtype and Ask2 enzyme from the lysine data in Fig. 2. The napp value for wildtype A K was 1.6, indicating a minimum of two lysine-binding sites on the active A K complex, which was similar to the Hill coefficient reported for A K from Black Mexican Sweet (Dotson et al. 1990). The navy value for homozy-
Table 1. Free and total amounts of aspartate-derived amino acids in whole kernels of wildtype (A619) and homozygous Ask2 mutant maize, expressed as nmol. (mg DW)- 1 Amino acid
1,0
84
0.8
Genotype
.~_ 0.6
Ask2/Ask2
Wildtype
Threonine Methionine Lysine Isoleucine
18 20 22 24 26 28 30 32 Fraction
Free
Total
Free
Total
.~ 0.4
0.10 0.07 0.13 0.09
28 15 15 28
17.4 0.7 1.7 0.20
47 27 20 28
t~ 0.2
The value for each genotype represents the mean of bulked 10kernel samples from each of five ears. Free amino acids were extracted into 5% trichloroacetic acid and total (free and proteinbound) amino acids were hydrolyzed in 6 N HC1 from two 500-mg portions of the same meal (Diedrick et al. 1990). Norleucine was included as an internal standard for amino-acid concentration determinations
0.(]
'
' 0.5
'
1.0 Log
~
1.5 (~M
-
":
2.0
2.5
=':= 3,0
"-
3.5
Lysine)
Fig. 2. Lysine inhibition of AKs purified from wildtype (e), heterozygous Ask (o), and homozygous Ask2 ( • ) maize. Relative activity was calculated for duplicate assays as Vi/Vo where Vo=activity at 0 lysine and Vi = activity at the given lysine concentration. Aspartate and MgATP were 10 and 5 mM, respectively, and lysine varied from 5 to 3 000 I-tM
S.B. Dotson et al. : Lysine-insensitive aspartate kinase in maize mutants 0.18
0.06
012
o04
549
09
~"0.06
c:: 0.02 -
13_ I-~0.04
~----0.03
~..0.02
~0,02 __.=
0
0.00 -40
/
0.04
0.06
Fig. 3. Replots of slope and intercept values versus lysine concentration for AK purified from wildtype A619 (o) and homozygous Ask2 (e). Slope and intercept values were obtained from primary doublereciprocal plots of I/V (pkat) versus 1/aspartate and 1/ATP. Aspartate concentrations varied (2, 2.5, 3.33, 5 and 10 mM) with ATP at 5 raM, and ATP concentrations varied (0.555, 0.714, l, 1.67 and 5 mM) with aspartate at 10 mM. Lysine was fixedchanging between 0 and 100 ~tM. Each slope and intercept line, representing single-assay data from a family of reciprocal plots; was calculated by leastsquares regression
0
40
80
0.01 120 -40 Lysine (gM)
i
0
40
gous mutant Ask2 A K was 0.7, indicating the loss of a lysine-binding site in the mutant enzyme 9 The biphasic lysine-inhibition curve of A K purified from heterozygous Ask plants indicated a mixture of wildtype and lysine-insensitive enzyme (Fig. 2). However, the lysine inhibition curve for homozygous Ask2 A K was not biphasic as would be expected for a mixture of lysine-sensitive A K expressed by the wildtype alleles at the Ask locus and lysine-insensitive enzyme expressed by the homozygous mutant alleles at the Ask2 locus if the two loci encoded discrete A K isoenzymes. Further analyses of A K were focused on comparisons of Ask2 and wildtype because the Ask2 enzyme appeared to be homogeneously altered to a lysine-insensitive form. The effects of 0 to 100 laM lysine on the relationships between velocity and varied MgATP and aspartate-substrate concentrations were examined for A K from homozygous Ask2. Slope and intercept values from doublereciprocal plots were replotted versus lysine concentration for wildtype and homozygous Ask2 (Fig. 3). As shown by the nearly horizontal lines for the homozygous Ask2 replots, lysine had little effect on substrate kinetics o f the mutant A K even at 100 gM which completely inhibited wildtype A K (Figs. 2, 3). The effects of lysine concentrations between 100 gM and 2500 gM on the homozygous Ask2 mutant A K also were investigated (Fig. 4). The double-reciprocal plots of 1/V versus 1/S were linear for both substrates, although there was some scatter in the points for the highest lysine concentration. Slope and intercept effects apparent from the graphical analysis o f 1/V versus 1/MgATP indicated linear noncompetitive inhibition. A non-competitive model was also favored over an uncompetitive model using regression analysis of the data set (Cleland 1979), as evidenced by the smallest standard errors for the kinetic constants (Table 2). Because A K catalyzes a two-substrate reaction, the apparent Ki constants with respect to ATP
OI
80
120
depend on the concentration of aspartate which was held constant at a nonsaturating level (10 mM). Ki values were at least 1.0 m M and were much higher than the minimum lysine concentration required for complete inhibition of wildtype A K (Table 2 A). Graphical analysis of velocity versus aspartate with lysine fixed-changing indicated that lysine was an uncompetitive inhibitor with respect to aspartate resulting in a Ki~nt) estimate of 0.9 m M lysine at 5 m M M g A T P (Table 2A, Fig. 4). The replots obtained from graphical analysis o f the slope and intercept values for both substrates were linear (Fig. 4, inset), indicating that lysine was a linear allosteric inhibitor of homozygous Ask2 mutant A K which was a distinct change from the parabolic inhibitor replots previously reported for wildtype A K (Dotson et al. 1990).
Substrate kinetics of AKfrom homozygous Ask2. Asparrate kinase from wildtype maize suspension-cell cultures was shown to catalyze a two-substrate, sequential reaction utilizing Mg2+ATP and aspartate (Dotson et al. 1990). The reaction kinetics o f A K from homozygous Ask2 were investigated to determine the effect o f the mutation reducing lysine inhibition on substrate kinetics. Double-reciprocal plots were linear, indicating lack of substrate cooperativity, and provided further evidence that Ask2 A K was kinetically homogeneous (Fig. 5). The possibility of a mixture of mutant and wildtype isoenzymes in extracts of homozygous mutant Ask2 plants with different substrate kinetics was further examined using Eadie-Scatchard plots which spread kinetic data at higher substrate concentrations further across the xaxis (Segal 1975). Eadie-Scatchard plots for A K from the homozygous Ask2 plants also were linear over a range o f substrate concentrations for both aspartate and ATP (data not shown). The intersecting lines for 1/V versus 1/ATP over a series of aspartate concentrations
550
S.B. Dotson et al. : Lysine-insensitive aspartate kinase in maize mutants
0.09 t0.04 10.02 f
0.06
t0.00
~
v-
-2000
Table 2. Estimated lysine inhibition constants (.4) and substrate kinetic constants (B) for AK purified from homozygous .4sk2 maize. Lysine inhibition constants were obtained by extrapolating replot data from Fig. 4 to the X-axis. Kinetic constants were obtained by fitting data from Fig. 5 to an equilibrium-ordered sequential model using the Eqordo computer program (Cleland 1979)
n
i
0
,
2000
_ . . ] ~ l z l
Lysine n ~
Constant a
Variable substrate (mM)
w
Aspartate 0.03
A Lysine [0.1-2.5 mM] Ki(slope) Ki(int)
0.00
'
'
'
'
0.0
'
'
0.4
0.8
t
0.04
_ ooo
~
1"
~X
oA
,,
oJ
t'-
1.2
1/Aspartate (mM-1) 0.09
ATP
.E "
/,/
1.1• 1.2•
0.9•
B Lysine absent 0.3•
Km(ATP) Ki(Asp)
21.9•
a Ki(slopeand int..... pt)= apparent inhibitor constants obtained by fitting ATP velocity data to a non-competitive model and the asparrate velocity data to an uncompetitive model: Km(ATP)=Michaelis constant for ATP with saturating aspartate; KicAsp)=dissociation constant for aspartate-enzyme complex
'' I
0.03
tD3
0.15
0.00
~
'
0.0
' 1.0
'
'
2.0
>
0.10
v
. . . .
o.o
1.o
6 e.o
~F'~
/47
w~
1/MgATP (mM-1)
I.L (2_ l---
0.05
Fig. 4. The effect of lysine on the relationship between velocity and substrate concentration for AK purified from homozygous Ask2 maize. Values of 1/V (pkat) were plotted versus 1/aspartate (0.91, 1.11, 1.43, 2, 3.33 and 10mM) at fixed 5mM ATP, and 1/ATP (0.45, 0.56, 0.71, 1, 1.67, and 5 mM) at fixed 10 mM aspartate in the presence of 0 (o), 0.1 (o), 0.5 ( x ) , 1.0 (,.) and 2.5 (D) mM lysine. The slope and intercept values for each family of data were calculated from a weighted fit of the reciprocal data (Cleland 1979) and replotted (insets) for each lysine concentration (0-2.5 mM). Extrapolation of the slope and intercept replots to the X-axis provided estimates of apparent lysine binding constants
0.00
0.0
0.4
1.2
0.8
1/Aspartate (mM -1)
0.15
- .('O
and 1/V versus 1/aspartate over a series of ATP concentrations indicated that the AK in the homozygous Ask2 mutant catalyzed an equilibrium-ordered sequential reaction mechanism (Cleland 1970). Furthermore, the family of lines for 1/V versus 1/ATP intersected on the vertical axis, indicating that aspartate bound to the enzyme before ATP. Replots of the slopes from the 1/V versus 1/aspartate plots (Slope 1/Asp) v e r s u s 1/ATP went through the origin (Fig. 5, inset) as expected for an equilibrium-ordered sequential mechanism (Cleland 1970). Michaelis and dissociation constants were calculated by fitting the data to a single equilibrium-ordered equation (Table 2B). The substrate kinetics of mutant Ask2 AK differed significantly from previously reported kinetic data for wildtype AK purified from suspension cultures which followed a non-equilibrium-ordered sequential mechanism (Dotson et al. 1990).
>
9
0.10
9
_..T--
O
o.o5
iV_
PI~LI"]_t~ 9 Springer-Verlag1990
Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize* Stanton B. Dotson**, David A. Frisch, David A. Somers***, and Burle G. Gengenbach Department of Agronomyand Plant Genetics,and Plant MolecularGenetics Institute, University of Minnesota, St. Paul, MN 55108, USA Received 10 May; accepted 7 June 1990
Abstract. Aspartate kinase (AK; EC 2.7.2.4) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations, AskLT19 and Ask2-LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived amino acids in maize. Kernels of the homozygous Ask2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine, compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 ~tM L-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygous Ask and homozygous Ask2 mutants required 25 and 760 gM lysine for 50% inhibition, respectively, indicating that Ask and Ask2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the homozygous mutant Ask2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutant Ask2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor of Ask2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive inhibition of wildtype AK. Reduced lysine sensitivity of the Ask2 gene product appeared to * Scientific paper No. 17419, Minnesota Agricultural Experiment Station projects No. 0302-4813-56 and No. 0302-4818-32 ** Present address: Monsanto Company, 700 ChesterfieldVillage Parkway, Chesterfield,MO 63198, USA *** To whom correspondence should be addressed Abbreviations: AK(s)= aspartate kinase(s); LT = equimolar lysine plus threonine
reduce the lysine inhibition of all of the AK activity detected in homozygous Ask2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from the Ask and Ask2 structural genes.
Key words: Amino acid biosynthesis Aspartate kinase - Lysine - Mutant (threonine overproduction) - Threonine - Zea (amino acids)
Introduction As the first enzyme in the biosynthesis pathway for the aspartate-family amino acids, aspartate kinase (AK; EC 2.7.2.4) potentially exerts a key regulatory role in the synthesis of lysine, threonine, methionine and other derivatives of the pathway (Bryan 1980). Aspartate kinase isoforms in plants are feedback-inhibited by lysine or threonine, and S-adenosyl methionine can increase the inhibition caused by low concentrations of lysine (Bryan 1980; Rognes et al. 1980). Growth of cell cultures and seedlings of many plants is inhibited by lysine plus threonine (LT), presumably because of feedback inhibition of AK, and possibly other steps in the pathway causing growth-limiting methionine biosynthesis levels (Dunham and Bryan 1969; Green and Phillips 1974; Gengenbach 1984). Selection for resistance to toxic LT levels, therefore, was proposed as a possible means to obtain mutations of AK that would be less sensitive to feedback regulation (Green and Phillips 1974). Mutants resistant to LT have been selected in maize (Zea mays L.) (Hibberd et al. 1980; Hibberd and Green 1982; Miao et al. 1988; Diedrick et al. 1990), carrot (Daucus carota L.) (Cattoir-Reynaerts et al. 1983), barley (Hordeum vulgare L.) (Bright et al. 1982) and tobacco (Nicotiana tabacum L.) (Bourgin et al. 1982). Increased accumulation of threonine in the free-amino-acid pool often is associated with LT resistance. In barley, LT-resistant
S.B. Dotson et al. : Lysine-insensitive aspartate kinase in maize mutants m u t a n t s were s h o w n to result f r o m t w o n o n a l l e l i c genes e n c o d i n g m u t a n t f o r m s o f A K i s o e n z y m e s t h a t were less sensitive to lysine f e e d b a c k i n h i b i t i o n ( A r r u d a et al. 1984; B r i g h t et al. 1982; R o g n e s et al. 1983). Several L T - r e s i s t a n t m a i z e lines h a v e been selected f r o m tissue c u l t u r e ( H i b b e r d et al. 1980; H i b b e r d a n d G r e e n 1982; M i a o et al. 1988; D i e d r i c k et al. 1990). T h e initially selected L T - r e s i s t a n t cell line h a d A K activity in c r u d e e x t r a c t s t h a t was a b o u t e i g h t f o l d less sensitive to lysine i n h i b i t i o n t h a n A K f r o m u n s e l e c t e d L T - s u s c e p tible c u l t u r e s ( H i b b e r d et al. 1980). P l a n t s r e g e n e r a t e d f r o m this c u l t u r e were infertile so p r o g e n y were n o t o b t a i n e d for genetic tests a n d the r e l a t i o n s h i p b e t w e e n alt e r e d A K a n d t h r e o n i n e o v e r p r o d u c t i o n c o u l d n o t be f u r t h e r investigated. S u b s e q u e n t l y , a d d i t i o n a l m a i z e m u t a n t s were selected for L T resistance a n d s h o w e d simple, d o m i n a n t , M e n d e l i a n i n h e r i t a n c e either for high freet h r e o n i n e level ( F r i s c h a n d G e n g e n b a c h 1986; D i e d r i c k et al. 1990) o r for g r o w t h o f seedlings ( H i b b e r d a n d G r e e n 1982) o r excised r o o t tips in the presence o f L T ( M i a o et al. 1988). F r e e - t h r e o n i n e c o n c e n t r a t i o n s in the m u t a n t k e r n e l s r a n g e f r o m 10-fold to m o r e t h a n 100-fold h i g h e r t h a n c o r r e s p o n d i n g w i l d t y p e kernels d e p e n d i n g o n the i n b r e d b a c k g r o u n d a n d allele c o m p o s i t i o n in the e m b r y o a n d e n d o s p e r m ( H i b b e r d a n d G r e e n 1982; M i a o e t a l . 1988). Recently, t w o t h r e o n i n e - o v e r p r o d u c i n g m a i z e p h e n o t y p e s were s h o w n to be c o n t r o l l e d b y n o n a l lelic loci, Ask a n d Ask21 ( F r i s c h a n d G e n g e n b a c h 1986; D i e d r i c k et al. 1990). T h e s e m u t a n t s o v e r p r o d u c e a s p a r t a t e - d e r i v e d a m i n o a c i d s ; h o w e v e r , the b i o c h e m i c a l basis for these m a i z e m u t a n t s h a s n o t yet been c h a r a c t e r i z e d . T h e objective o f this s t u d y was to c h a r a c t e r i z e the A K a c t i v i t y f r o m the t w o L T - r e s i s t a n t m u t a n t m a i z e g e n o t y p e s , in o r d e r to u n d e r s t a n d the role o f this e n z y m e in r e g u l a t i n g the synthesis o f a s p a r t a t e - d e r i v e d a m i n o acids. L y s i n e - i n s e n s i t i v e f o r m s o f A K c o s e g r e g a t e d w i t h a m i n o - a c i d o v e r p r o d u c t i o n in b o t h m a i z e m u t a n t s , est a b l i s h i n g t h a t o v e r p r o d u c t i o n was c a u s e d b y m u t a t i o n s in the t w o A K s t r u c t u r a l loci. W e were able to derive a line h o m o z y g o u s for the Ask2 m u t a t i o n . P u r i f i e d Ask2 A K w a s h o m o g e n e o u s l y lysine-insensitive i n d i c a t i n g t h a t the g e n e - e n z y m e r e l a t i o n s h i p s o f m a i z e A K likely differed f r o m the discrete i s o e n z y m e c o m p o s i t i o n des c r i b e d for A K in b a r l e y ( A r r u d a et al. 1984; B r i g h t et al. 1982; R o g n e s et al. 1983). F u r t h e r m o r e , f e e d b a c k insensitive f o r m s o f A K c o n f e r r e d s u b s t a n t i a l o v e r p r o d u c t i o n o f t h r e o n i n e , lysine, m e t h i o n i n e a n d isoleucine, i n d i c a t i n g e l e v a t e d flux t h r o u g h the p a t h w a y a n d d e m o n s t r a t i n g t h a t A K p l a y s a k e y r e g u l a t o r y role for the p a t h w a y in maize.
Material and methods Mutant and wildtype genotypes. Maize (Zea mays L.) inbred line A619 served as the wildtype control and was used as the recurrent Locus and allele designations conform to guidelines for maize genetic nomenclature (Burnham et al. 1975) in which the first locus of a series is given a three-letter designation indicative of the phenotype (Ask for aspartate kinase) and succeeding loci are designated by the same letters plus a number (Ask2)
547 parent in developing the derivative mutant genotypes following selection for LT resistance in tissue culture. The mutant allele (AskLT19) for one AK (Ask) locus was derived from LT-resistant LT19 plants produced as described by Hibberd and Green (1982). Plants containing the Ask-LT19 allele (provisionally designated Ltr*19 by Hibberd and Green 1982) were backcrossed seven generations to wildtype A619 plants and then progeny were self-pollinated two generations to produce a limited amount of A619Ask-LT19/AskLT19 seed. Because few homozygous Ask seed were available, a uniform population of heterozygous A619Ask-LT19/+ plants was obtained by a final cross to A619 for the purpose of enzyme analysis. The mutant allele (Ask2-LT20) of the second AK locus (Ask2) was obtained from plants regenerated from another tissue culture selected for LT resistance (Diedrick et al. 1990). A homozygous mutant line of A619Ask2-LT20/Ask2-LT20 was obtained after backcrossing five generations to A6194./4-;+/4- followed by three generations of selfing. To simplify genotype designations throughout the remainder of this report, the genotypes with respect to the Ask and Ask2 loci are as follows: wildtype=A619 4-/4-; 4-/4- ; heterozygous Ask=A619Ask-LT19/ 4- ; 4-/4- ; and homozygous Ask2=A6194-/4-;Ask2-LT20/Ask2-LT20. These mutant genotypes were confirmed by analyses of test crosses to wildtype A619 in 1988. Seed supplies of both mutant lines were not adequate for enzyme extraction from seedlings; therefore, plants were grown to anthesis in the field in 1988 and enzyme extracted from immature ears. Also, since the experimental hypothesis was that increased kernel threonine was the result of an altered AK, it made sense to extract AK from a related tissue such as immature ears. Unpollinated, immature ears (3-8 cm) were removed from the husks and silks, quickly frozen and ground in liquid nitrogen using a stainless steel Waring blender, and stored under nitrogen at - 7 0 ~ C until enzyme purification.
Enzyme purification. Aspartate kinase from immature ears was extracted and purified as described for maize suspension-cell cultures (Dotson et al. 1989). Crude extracts were desalted, and partially purified AK was obtained by successive chromatographic steps on phenyl sepharose hydrophobic, Sephacryl S300HR gel filtration, and FPLC Mono Q anion-exchange columns. The buffers used for anion exchange were prepared in a single batch and used during the purification of AK from each genotype to allow comparison of elution profiles. Several purified preparations of AK from each source were combined (approx. 600 g FW total) to obtain enough enzyme for kinetic analyses. After ion-exchange chromatography, 2 mg/ml bovine serum albumin was added to each preparation to stabilize enzyme activity, and stored at - 7 0 ~ C. Aspartate-kinase assays. A modified aspartate-hydroxamate method was used during enzyme purification and the pyruvate kinaselactate dehydrogenase coupled assay was used for kinetic analysis (Dotson et al. 1989, 1990). Amino-acid analysis. Free and total concentrations of amino acids in kernels harvested from wildtype and homozygous Ask2 plants were extracted and analyzed as previously described (Diedrick et al. 1990). Data analysis. Aspartate-dependent AK activity was expressed in pkat (pmol-s -1) and analyzed according to graphical methods (Cleland 1967, 1970). Computer programs Eqordo and Hypero (Cleland 1979) were supplied by Charles B. Grissom (Department of Chemistry, University of California, Berkeley, USA) and used to fit substrate kinetic data and hyperbolic velocity data for lysine inhibition, respectively. The programs Uncomp, Comp and Noncomp were used as additional evidence to discriminate an uncompetitive versus a non-competitive inhibition and to derive the inhibition constants in Table 2. Lysine inhibition data also were fitted to the logarithmic Hill equation (Segal 1975) by plotting log[V/ (Vm.x-V)] versus log [lysine].
548
S.B. Dotson et al. : Lysine-insensitive aspartate kinase in maize mutants
Results
,.-, 3.0
Amino-acid composition. The free and total amounts of aspartate-derived amino acids in wildtype and homozygous m u t a n t Ask2 kernels are shown in Table 1. Free threonine, methionine, lysine, and isoleucine were increased 174-, 10-, 13-, and 2-fold, in the order given in the m u t a n t kernels c o m p a r e d to wildtype. The increased free threonine contributed to a significant increase in total kernel threonine. Increases in total methionine and lysine were associated with, but not fully attributable to the free-pool increases, indicating proteinb o u n d methionine or lysine composition also changed. No significant differences in total kernel composition were observed in other amino acids except for a 10-fold increase in free serine (data not shown). Heterozygous Ask kernels were previously shown to have a 29-fold increase in free threonine c o m p a r e d to wildtype (Hibberd and Green 1982). Enzyme purification. Aspartate kinase was partially purified f r o m immature ears o f wildtype, heterozygous Ask and homozygous Ask2 plants using a procedure that was developed for A K purification from Black Mexican Sweet maize suspension-culture cells and routinely resulted in 1200-fold purification with greater than 85% recovery (Dotson et al. 1989). Specific activities in desalted crude extracts from the immature ears of wildtype and the mutants were approx. 14 p k t a l . ( m g protein)-1 indicating that the increased threonine in the kernel was not the result of A K overexpression. Aspartate-kinase activity f r o m the three genotypes eluted from a Sephacryl S300HR gel-filtration column as single peaks of Mr about 254000 indicating nearly identical sizes. The A K activity from wildtype and homozygous Ask2 eluted f r o m the M o n o Q anion-exchange column as single peaks at similar positions in the elution gradient (Fig. 1). Aspartate kinase f r o m heterozygous Ask plants eluted as an early b r o a d shoulder followed by a major activity peak, presumably indicating a mixture of wildtype and
_~_~c2 . 0 v
._> 1.o < 0.0
Anion-exchange chromatography elution of AK activity from immature ears of wildtype A619, heterozygous Ask, and homozygous Ask2 maize. A Fast Protein Liquid Chromatography Mono Q column was eluted with a linear 75-275 mM KC1 gradient. o, Wildtype A619; o, Ask/4-; x , Ask2/Ask2 Fig. l.
mutant A K isoforms. Characterization of the major peak of A K purified from the Ask plants is reported herein.
Lysine inhibition of AK activities &olated from Ask and Ask2. Approximately 10 ~tM lysine was required to inhibit 50% of the A K activity 05o) from wildtype maize as determined from fit to the Hill equation (Fig. 2). In contrast, the I5o values for the A K activity from heterozygous Ask and homozygous Ask2 plants were 25 laM and 760 laM lysine, respectively. These results indicated that the amino-acid overproduction of the mutations is conferred by a reduction in lysine inhibition o f AK. Therefore, Ask and Ask2 appear to be independent loci encoding lysine-regulated subunits of AK. Hill coefficients were calculated for the wildtype and Ask2 enzyme from the lysine data in Fig. 2. The napp value for wildtype A K was 1.6, indicating a minimum of two lysine-binding sites on the active A K complex, which was similar to the Hill coefficient reported for A K from Black Mexican Sweet (Dotson et al. 1990). The navy value for homozy-
Table 1. Free and total amounts of aspartate-derived amino acids in whole kernels of wildtype (A619) and homozygous Ask2 mutant maize, expressed as nmol. (mg DW)- 1 Amino acid
1,0
84
0.8
Genotype
.~_ 0.6
Ask2/Ask2
Wildtype
Threonine Methionine Lysine Isoleucine
18 20 22 24 26 28 30 32 Fraction
Free
Total
Free
Total
.~ 0.4
0.10 0.07 0.13 0.09
28 15 15 28
17.4 0.7 1.7 0.20
47 27 20 28
t~ 0.2
The value for each genotype represents the mean of bulked 10kernel samples from each of five ears. Free amino acids were extracted into 5% trichloroacetic acid and total (free and proteinbound) amino acids were hydrolyzed in 6 N HC1 from two 500-mg portions of the same meal (Diedrick et al. 1990). Norleucine was included as an internal standard for amino-acid concentration determinations
0.(]
'
' 0.5
'
1.0 Log
~
1.5 (~M
-
":
2.0
2.5
=':= 3,0
"-
3.5
Lysine)
Fig. 2. Lysine inhibition of AKs purified from wildtype (e), heterozygous Ask (o), and homozygous Ask2 ( • ) maize. Relative activity was calculated for duplicate assays as Vi/Vo where Vo=activity at 0 lysine and Vi = activity at the given lysine concentration. Aspartate and MgATP were 10 and 5 mM, respectively, and lysine varied from 5 to 3 000 I-tM
S.B. Dotson et al. : Lysine-insensitive aspartate kinase in maize mutants 0.18
0.06
012
o04
549
09
~"0.06
c:: 0.02 -
13_ I-~0.04
~----0.03
~..0.02
~0,02 __.=
0
0.00 -40
/
0.04
0.06
Fig. 3. Replots of slope and intercept values versus lysine concentration for AK purified from wildtype A619 (o) and homozygous Ask2 (e). Slope and intercept values were obtained from primary doublereciprocal plots of I/V (pkat) versus 1/aspartate and 1/ATP. Aspartate concentrations varied (2, 2.5, 3.33, 5 and 10 mM) with ATP at 5 raM, and ATP concentrations varied (0.555, 0.714, l, 1.67 and 5 mM) with aspartate at 10 mM. Lysine was fixedchanging between 0 and 100 ~tM. Each slope and intercept line, representing single-assay data from a family of reciprocal plots; was calculated by leastsquares regression
0
40
80
0.01 120 -40 Lysine (gM)
i
0
40
gous mutant Ask2 A K was 0.7, indicating the loss of a lysine-binding site in the mutant enzyme 9 The biphasic lysine-inhibition curve of A K purified from heterozygous Ask plants indicated a mixture of wildtype and lysine-insensitive enzyme (Fig. 2). However, the lysine inhibition curve for homozygous Ask2 A K was not biphasic as would be expected for a mixture of lysine-sensitive A K expressed by the wildtype alleles at the Ask locus and lysine-insensitive enzyme expressed by the homozygous mutant alleles at the Ask2 locus if the two loci encoded discrete A K isoenzymes. Further analyses of A K were focused on comparisons of Ask2 and wildtype because the Ask2 enzyme appeared to be homogeneously altered to a lysine-insensitive form. The effects of 0 to 100 laM lysine on the relationships between velocity and varied MgATP and aspartate-substrate concentrations were examined for A K from homozygous Ask2. Slope and intercept values from doublereciprocal plots were replotted versus lysine concentration for wildtype and homozygous Ask2 (Fig. 3). As shown by the nearly horizontal lines for the homozygous Ask2 replots, lysine had little effect on substrate kinetics o f the mutant A K even at 100 gM which completely inhibited wildtype A K (Figs. 2, 3). The effects of lysine concentrations between 100 gM and 2500 gM on the homozygous Ask2 mutant A K also were investigated (Fig. 4). The double-reciprocal plots of 1/V versus 1/S were linear for both substrates, although there was some scatter in the points for the highest lysine concentration. Slope and intercept effects apparent from the graphical analysis o f 1/V versus 1/MgATP indicated linear noncompetitive inhibition. A non-competitive model was also favored over an uncompetitive model using regression analysis of the data set (Cleland 1979), as evidenced by the smallest standard errors for the kinetic constants (Table 2). Because A K catalyzes a two-substrate reaction, the apparent Ki constants with respect to ATP
OI
80
120
depend on the concentration of aspartate which was held constant at a nonsaturating level (10 mM). Ki values were at least 1.0 m M and were much higher than the minimum lysine concentration required for complete inhibition of wildtype A K (Table 2 A). Graphical analysis of velocity versus aspartate with lysine fixed-changing indicated that lysine was an uncompetitive inhibitor with respect to aspartate resulting in a Ki~nt) estimate of 0.9 m M lysine at 5 m M M g A T P (Table 2A, Fig. 4). The replots obtained from graphical analysis o f the slope and intercept values for both substrates were linear (Fig. 4, inset), indicating that lysine was a linear allosteric inhibitor of homozygous Ask2 mutant A K which was a distinct change from the parabolic inhibitor replots previously reported for wildtype A K (Dotson et al. 1990).
Substrate kinetics of AKfrom homozygous Ask2. Asparrate kinase from wildtype maize suspension-cell cultures was shown to catalyze a two-substrate, sequential reaction utilizing Mg2+ATP and aspartate (Dotson et al. 1990). The reaction kinetics o f A K from homozygous Ask2 were investigated to determine the effect o f the mutation reducing lysine inhibition on substrate kinetics. Double-reciprocal plots were linear, indicating lack of substrate cooperativity, and provided further evidence that Ask2 A K was kinetically homogeneous (Fig. 5). The possibility of a mixture of mutant and wildtype isoenzymes in extracts of homozygous mutant Ask2 plants with different substrate kinetics was further examined using Eadie-Scatchard plots which spread kinetic data at higher substrate concentrations further across the xaxis (Segal 1975). Eadie-Scatchard plots for A K from the homozygous Ask2 plants also were linear over a range o f substrate concentrations for both aspartate and ATP (data not shown). The intersecting lines for 1/V versus 1/ATP over a series of aspartate concentrations
550
S.B. Dotson et al. : Lysine-insensitive aspartate kinase in maize mutants
0.09 t0.04 10.02 f
0.06
t0.00
~
v-
-2000
Table 2. Estimated lysine inhibition constants (.4) and substrate kinetic constants (B) for AK purified from homozygous .4sk2 maize. Lysine inhibition constants were obtained by extrapolating replot data from Fig. 4 to the X-axis. Kinetic constants were obtained by fitting data from Fig. 5 to an equilibrium-ordered sequential model using the Eqordo computer program (Cleland 1979)
n
i
0
,
2000
_ . . ] ~ l z l
Lysine n ~
Constant a
Variable substrate (mM)
w
Aspartate 0.03
A Lysine [0.1-2.5 mM] Ki(slope) Ki(int)
0.00
'
'
'
'
0.0
'
'
0.4
0.8
t
0.04
_ ooo
~
1"
~X
oA
,,
oJ
t'-
1.2
1/Aspartate (mM-1) 0.09
ATP
.E "
/,/
1.1• 1.2•
0.9•
B Lysine absent 0.3•
Km(ATP) Ki(Asp)
21.9•
a Ki(slopeand int..... pt)= apparent inhibitor constants obtained by fitting ATP velocity data to a non-competitive model and the asparrate velocity data to an uncompetitive model: Km(ATP)=Michaelis constant for ATP with saturating aspartate; KicAsp)=dissociation constant for aspartate-enzyme complex
'' I
0.03
tD3
0.15
0.00
~
'
0.0
' 1.0
'
'
2.0
>
0.10
v
. . . .
o.o
1.o
6 e.o
~F'~
/47
w~
1/MgATP (mM-1)
I.L (2_ l---
0.05
Fig. 4. The effect of lysine on the relationship between velocity and substrate concentration for AK purified from homozygous Ask2 maize. Values of 1/V (pkat) were plotted versus 1/aspartate (0.91, 1.11, 1.43, 2, 3.33 and 10mM) at fixed 5mM ATP, and 1/ATP (0.45, 0.56, 0.71, 1, 1.67, and 5 mM) at fixed 10 mM aspartate in the presence of 0 (o), 0.1 (o), 0.5 ( x ) , 1.0 (,.) and 2.5 (D) mM lysine. The slope and intercept values for each family of data were calculated from a weighted fit of the reciprocal data (Cleland 1979) and replotted (insets) for each lysine concentration (0-2.5 mM). Extrapolation of the slope and intercept replots to the X-axis provided estimates of apparent lysine binding constants
0.00
0.0
0.4
1.2
0.8
1/Aspartate (mM -1)
0.15
- .('O
and 1/V versus 1/aspartate over a series of ATP concentrations indicated that the AK in the homozygous Ask2 mutant catalyzed an equilibrium-ordered sequential reaction mechanism (Cleland 1970). Furthermore, the family of lines for 1/V versus 1/ATP intersected on the vertical axis, indicating that aspartate bound to the enzyme before ATP. Replots of the slopes from the 1/V versus 1/aspartate plots (Slope 1/Asp) v e r s u s 1/ATP went through the origin (Fig. 5, inset) as expected for an equilibrium-ordered sequential mechanism (Cleland 1970). Michaelis and dissociation constants were calculated by fitting the data to a single equilibrium-ordered equation (Table 2B). The substrate kinetics of mutant Ask2 AK differed significantly from previously reported kinetic data for wildtype AK purified from suspension cultures which followed a non-equilibrium-ordered sequential mechanism (Dotson et al. 1990).
>
9
0.10
9
_..T--
O
o.o5
iV_
