A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure

72

Biochimica et Biophysica Acta 913 (1987) 72 80

Elsevier BBA 32829

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure

A n t h o n y R. Clarke a, Dale B. Wigley a, David A. Barstow b, William N. Chia a, Tony Atkinson b and J. John Holbrook a Department of Biochemistry, University of Bristol Medieal School, University Walk, Bristol and h Microbial Technology Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury (U. K.)

(Received 20 October 1986)

Key words: Site-directed mutagenesis; Lactate dehydrogenase; Protein engineering; Allosteric regulation; Oligomeric stability; (Bacteria)

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-bisphosphate (Fru-l,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (K d NADH, K m pyruvate, K i oxamate and kc,t) , but has weakened the binding of Fru-l,6-P 2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-l,6-P2, is important for ailosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.

Introduction M a n y lactate dehydrogenase from prokaryotic sources are allosterically activated by the glycolytic intermediate F r u - l , 6 - P 2 [1-5]. This regulation is metabolically significant in that the conversion of pyruvate to lactate is critical in determining the fate of carbon sources in bacterial fermentation (for review see Ref. 6). When the enzyme is active, glucose is converted to lactate, Abbreviation: LDH~ lactate dehydrogenase. Corresponding: A.R. Clarke, Department of Biochemistry, Medical School, University Walk, Bristol BS8 ITD, U.K.

thus regenerating N A D + to sustain the glycolytic pathway; when inactive, other fermentation products are generated (eg., acetate, formate, carbon dioxide and ethanol). Results with streptococcal bacteria [7,8] have shown that in conditions of excess glucose there is a raised level of intracellular F r u - l , 6 - P 2 which leads to a 'feed-forward' stimulation of lactate dehydrogenase and lactate is secreted as the only fermentation product. However, when glucose is limiting in the growth medium, the intracellular Fru-l,6-P 2 concentration is undetectably small and 90% of the carbon source is converted to acetate (despite the organism's being traditionally considered a homolactic fermenter).

0167-4838/87/$03.50 ~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)

73

The best understood Fru-l,6-Pz-sensitive lactate dehydrogenase is that from Bacillus stearothermophilus; there is both an amino acid [9,10] and a gene sequence [11] available and much is known about its kinetic, ligand-binding and quaternary structural properties [1,12-4]. Additionally, we have produced an Escherichia coli clone which produces 25 30% of its soluble protein as B. stearothermophilus lactate dehydrogenase from a gene inserted behind the TAC promoter in the pKK223-3 plasmid [11]. We have studied in some detail the molecular mechanism by which Fru-l,6-P 2 activates this enzyme [12-14] and our major findings are that the enzyme exists as a dimer and a tetramer, with Fru-l",6-P2 binding weakly to the former ( K d 3 mM) and tightly to the latter ( K d 6 8 ~M). The binding of N A D H is not effected by either protein-protein association or Fru-l,6-P2-binding, but the binding of substrate is tightened 50-100-fold when the dimer is converted to the Fru-l,6-P2-tetramer complex. This improvement in affinity occurs in two steps; the binding of Fru-l,6-P 2 to the dimer alone accounts for an 8-fold increase, and the dimer-to-tetramer conversion for a 10-15-fold increase. Thus adding Fru-l,6-P 2 to a dilute solution of the protein both promotes tetramer formation and alters the K m for pyruvate from 2 - 3 mM to 30-40/~M. Eukaryotic lactate dehydrogenases have a distinct anion-binding site on each polypeptide chain, close to a subunit interface [15], and it has been suggested that Fru-l,6-P 2 binds to an homologous site in prokaryotic lactate dehydrogenases [13,14, 16]. This proposal is based, firstly, on the structural similarity between prokaryotic and eukaryotic lactate dehydrogenases [9], secondly, on the experimental observation by Hensel et al. [17] that chemical modification of His-188 (a residue at the anion site in eukaryotes) abolishes the effect of Fru-l,6-P 2 on the enzyme from Bacillus caldotenax, and thirdly, on a preliminary structure of the Lactobacillus casei L D H - F B P complex (Buehner, M., personal communication). We have fitted the amino acid sequence of B. stearothermophilus lactate dehydrogenase into the X-ray coordinates of the homologous pig H 4 enzyme (the L D H S-lac-NAD complex [18]) and the structure shows that there are two positively charged residues at

this anion site (which it is suggested accommodates one of the two phosphates of Fru-l,6-P2). One is His-188 and the other Arg-173. These two residues are components of two pieces of secondary structure - the a-2F helix (residues 163-179) and the fiG~fill extended chain (182-196). These elements of secondary structure extend over 20 ,~ through the core of the subunit and at their other end form the substrate-binding site. In a previous paper [14], we suggested that the binding of a phosphate group on Fru-l,6-P z between Arg-173 and His-188 will alter the relative position of the a-2F and f i G / f i l l polypeptide chains and allow communication with the active site of the enzyme. In the experiments described in this paper we have used used site-directed mutagenesis to replace Arg-173 by glutamine in order to test the above hypothesis and to establish the general role of this residue in determining the quaternary structure and allosteric activation of the enzyme. Materials and Methods

Mutagenesis. A single, point mutation ( C C G CAG)

was

introduced

into

the

cloned

B.

stearothermophilus lactate dehydrogenase gene (see Ref. 11) at the codon for residue 173, so that Arg 'was replaced by Gin. This maniputation was accomplished by the oligonucleotide-mismatch procedure in the M13 mp8 vector as described by Winter et al. [19] using a 22-base mismatch oligonucleotide as the primer for chain extension. Enzyme purification. The mutated gene was expressed in E. coli by insertion into the pKK2233 plasmid behind the TAC promoter [11]. Of the soluble protein in culture grown overnight in a 2 × Y T medium, 20% was the mutant B. stearothermophilus lactate dehydrogenase. The enzyme was purified as described previously for the wild-type enzyme [12] except that sodium chloride and Fru-l,6-P 2 were omitted from the buffers used in the affinity purification step on oxamateSepharose (the enzyme was found not to be activated by Fru-l,6-P 2 in a crude-cell extract and the salt concentration was lowered to compensate for the consequently lower substrate affinity). The isolated enzyme was judged better than 98% pure on polyacrylamide gel-electrophoresis in SDS [20]. Fluorescence polarization measurements. The

74 m u t a n t lactate d e h y d r o g e n a s e was covalently labelled with the fluorescent dye, fluorescamine, as described previously [12]. The d y e - e n z y m e conj u g a t e h a d the same specific activity as the u n l a b e l l e d enzyme. M e a s u r e m e n t s of the equil i b r i u m fluorescence-polarization of this conjugate a n d of N A D H were m a d e as described in Ref. 13.

Measurements of total fluorescence intensity. These were m a d e on an S L M p h o t o n counting s p e c t r o f l u o r i m e t e r ( S L M Instruments, U r b a n a , IL, U S A ) with m o n o c h r o m a t i o n of b o t h exciting a n d e m i t t e d light. Buffers and temperature. Unless otherwise stated, all e x p e r i m e n t s were carried out at 2 5 ° C in buffers c o n t a i n i n g 20 m M t r i e t h a n o l a m i n e H C I , / N a O H ( p H 6.0).

Results (1) The effect of the mutation on the quaternary structure of the enzyme. W h e n the m u t a n t enzyme is assayed at a p y r u vate c o n c e n t r a t i o n below the K m (0.1 m M ) a n d in the absence of F r u - l , 6 - P z, the o r d e r of m i x i n g of the assay c o m p o n e n t s (enzyme, N A D H a n d pyruvate) has a n o t i c e a b l e effect on the progress curve of the reaction (see Fig. 1). If the reaction is 1.25 --

1.20


1.15

1.10

;

do Time

,oo

1;o

(s)

Fig. 1. The effect of mixing order on the progress curve for pyruvate reduction. Two reactions were carried out with the mutant enzyme with the following concentrations of components in a 2.5 ml cuvene; 0.2 mM NADH/0.1 mM pyruvate/0.01 /~M enzyme sites. In one case (a), the reaction was initiated by the addition of 12.5/d of 2 ~M enzyme (sites), and in the other (b), by the addition of 25 ~1 of 20 mM NADH. The reaction was followed by measuring the decrease in NADH concentration reflected by the decrease in A340.

initiated b y the a d d i t i o n of N A D H , a steady r e a c t i o n rate is achieved i m m e d i a t e l y a n d the progress curve is near-linear (there is a very slight acceleration) until a significant p r o p o r t i o n of the p y r u v a t e has been consumed. However, when an otherwise identical reaction is initiated b y the a d d i t i o n of enzyme, the reaction rate - over the s a m e time-course - is initially much faster a n d decays over a p e r i o d of 2 - 3 min to a p p r o x i m a t e l y the same rate as seen when N A D H starts the reaction. O n e of m a j o r differences b e t w e e n these reactions is that in the former the enzyme conc e n t r a t i o n at initiation is very low (equilibrated in the cuvette at 0.005 /~M), and in the latter relatively high (2.00 /~M before dilution into the cuvette). In the w i l d - t y p e enzyme there is no difference in the progress curve for these two assays because the p r o t e i n is p r e d o m i n a n t l y dimeric at b o t h these p r o t e i n c o n c e n t r a t i o n s [13] a n d so w o u l d show the kinetic characteristics of this form of the enzyme. This need not be the case for the m u t a n t enzyme and, indeed, the a b o v e observation suggested to us that between the two conc e n t r a t i o n s of enzyme there is a transition between forms with different kinetic characteristics. Hence, when the reaction is started with enzyme, there m a y be a slow subunit dissociation which is reflected in the decay in the reaction rate (see section 4 for an analysis of this effect). W e investigated this possible change in the stability of q u a t e r n a r y states of the enzyme through m e a s u r e m e n t s of m o l e c u l a r size by fluorescence polarization. Fluorescamine-labelled m u t a n t lactate d e h y d r o g e n a s e was equilibrated at a c o n c e n t r a t i o n of 0.05 ~ M (in terms of dimers) a n d unlabelled enzyme was a d d e d to the cuvette. T h e response of the H / V ratio is shown in Fig. 2A. The results show that an increasing p r o t e i n c o n c e n t r a t i o n leads to an increase in the size of the labelled particle which manifests itself as a decrease in the H / V ratio (see Ref. 13). The analysis of the result (Fig. 2(B)) by the graphic p r o c e d u r e derived previously for the w i l d - t y p e e n z y m e [13] yields a dissociation c o n s t a n t for a simple A + A = B interaction of 0.08 ~ M ( K d for the w i l d - t y p e enzyme is 33.00 /~M). F u r t h e r m o r e , the H / V ratio for the species at high p r o t e i n c o n c e n t r a t i o n is 0.515, while the e x t r a p o l a t e d value for the species p r e s e n t at an infinitely low con-

75

0.515

0.50

1.0

4

k.

A

\

%v o.55c~

0.5 cx

A

J

0"5850

2

4

0

O~ 0.25

0

..

,

q;)

,

•

.00"

-.

¢i~i)'~. -

-

_

s

I

10

0

2 [d]mer] 0 ~M

I

,'s

2C)

25

[FBP] mM

5O

y-/ B

O( (1-002

O~ 2 (1- OLl 25

i

O0-

_

_2

4 0

2 [d;Mer] o pM Fig. 2. The subunit assembly on raising protein concentration. (A) Aliquots of concentrated, unlabelled lactate dehydrogenase were added to a 2.5 ml solution of 0.05 /*M (dimer) fluorescamine-labelled lactate dehydrogenase and the ratio of the horizontal to vertical fluorescence intensities ( H / V ) was measured (after equilibration) as the enzyme concentration was increased. [dimer]o is the total enzyme concentration expressed as dimers. (B) The results were plotted according to the equation: o~/(1 - a) 2 = 2 [ d i m e r ] o / K p, where Kp is the dimer-dimer dissociation constant and c~ is the proportion of enzyme in the tetrameric form (see text and Ref. 13).

0

i

i

I

2

i

3

2 [dimer]0 )uM

Fig. 3. The influence of Fru-l,6-P 2 on the stability of the tetramer. (A) Aliquots of Fru-l,6-P 2 were added to labelled lactate dehydrogenase (0.05 ~tM dimer) and the H~ V ratio of the extrinsic fluorescence was recorded at equilibrium. Superimposed on the experimental results are three simulated curves generated by a model which assumes appreciable binding only to the dimeric form of the enzyme. +2F

T ~- 2D ,~ 2DF

centration of protein is 0.585. These values are extremely close to those measured for the wild-type enzyme (0.510 and 0.590, respectively [13]), where it has been shown that the equilibrium is between the dimer and the tetramer. This experiment demonstrates that the mutant enzyme undergoes the same change in subunit assembly as the wild-type, but that its tetrameric form is 400-times more stable. (2) The influence of Fru-l,6-P2 on the dimer-tetramer equilibrium Fig. 3A shows which

the result of an experiment

F r u - l , 6 - P 2 is a d d e d

rescamine-labelled

enzyme.

to a solution The

in

of fluo-

enzyme

con-

analytical solution: 2D

(1

- - 0~) 2

-1

where F, D and T are Fru-l,6-P 2, dimeric lactate dehydrogenase and tetrameric lactate dehyrogenase, respectively, a is the proportion of protein in the tetrameric form and K r is the dissociation constant for the Fru-l,6-P2-dimer interaction. For the simulations we used the measured value for Kp (0.08/*M) and the three curves (i, ii and iii) are generated by assigning Kr values of 15 mM, 8 m M and 5 mM, respectively. (B) After the addition of 21 m M Fru-l,6-P 2 the titration was stopped and unlabelled protein was added to the cuvette. The H~ V ratio was then recorded at increasing protein concentrations (as in the experiment described in Fig. 2). Shown here is the transform of these data which yields an apparent Kp of 1 ttM.

76

centration is such that the dimer-tetramer equilibrium is poised at around its mid-point. Addition of F r u - l , 6 - P 2 to this solution leads not to an assembly of the protein as it does in the wild-type [13], but to a dissociation to dimers. At an F r u - l , 6 - P 2 concentration of 21 m M this process is almost complete and we conclude that the molecule must bind weakly to the dimer, but even more weakly or not at all to the tetramer. If it is assumed that appreciable binding occurs only to the dimer, then analysis of the data using the F A C S I M I L E algorithm [21] yields a value for K r of 8 m M (see Fig. 2A and legend). This result was confirmed by addition of unlabelled enzyme to the same cuvette (i.e., in the presence of 21 m M Fru-l,6-P2) to determine the affinity between dimers in these conditions (see Fig. 3B). We find, as expected, that the apparent K d has increased, showing that the tetramer forms less easily F r u - l , 6 - P 2 is present. The observed value for the apparent dissociation constant is 1.00 /~M and the predicted value from the model described in the legend is 1.05/~M.

(3) The catalytic consequences of the mutation Figs. 4A and B show double-reciprocal plots for assays in which the pyruvate concentration was varied. The plot shown in Fig. 4A is for the dimeric form of the enzyme with and without F r u - l , 6 - P 2, the rates being measured in assays initiated by the addition of N A D H , so that the enzyme was equilibrated at a low concentration (0.005 /~M dimer) and was dissociated. The plot shown in Fig. 4B compares the characteristics of the tetramer with those of the dimer. For the former the reaction was initiated by the addition of enzyme from a 1.00 ktM stock so that the enzyme started as a tetramer and slowly dissociated during the reaction (see section 4). The dissociation is sufficiently slow that measurement of the rate over the first 5 s will yield the kinetic features of the tetrameric form. The results show that the dimer in the absence of F r u - l , 6 - P 2 has a K,1, for pyruvate of 1.6-2.0 m M and a k~, t of 230-260 s - I ; these values are very close to those determined for the wild-type enzyme (2 3 m M and 250 s -1 [12,13]. Addition of Fru-l,6-P 2, however, rather than stimulating the dimer as in the wild-type enzyme [4], acts as a

30

20

>

10

o

-2'.5

/

A

2'.s Es}-lmM1

51o

3C

E

B

4

10

-215

0

2'.s is] -1 raM-1

5'.o

Fig. 4. Steady-state characteristics of the mutant enzyme. The double-reciprocal plots shown in (A) and for the dimeric enzyme in the presence of 20 mM Fru-l,6-P 2 (D), and in its absence (I). Cuvettes contained 0.005 ~M lactate dehydrogenase sites and varying concentrations of pyruvate. The reactions were initiated by addition of a saturating concentration of N A D H (0.2 mM). Those shown in (B) directly compare the tetramer (A), and the dimer (I), each in the absence of Fru-I ~61"2. For the former the reaction was initiated by the addition of a small aliquot of concentrated enzyme (2 btM sites), but otherwise conditions were exactly as in (A). All rates were determined by measuring the change in A3,~).

mild inhibitor over the measured range, reducing t h e k c a , to 1 3 0 s ~ and t h e K m to 0 . 8 m M . I n t h e wild-type enzyme, F r u - l , 6 - P 2 activation of the dimer is very marked and leads to a reduction in the pyruvate K m of at least 7-fold [14]. The formation of the mutant tetramer, however (see Fig. 4B), activates the enzyme slightly at concentrations of substrate up to 1.0 mM. The K m and kca t for this species are 0.65 m M and 170 s ~, respectively; this is analogous to the improvement in the affinity of oxamate binding in the tetramer (in the absence of F r u - l , 6 - P 2) which is a characteristic of both the mutant and wild-type enzyme (see section 6).

77

(4) Measurement of the rate of tetramer-to-dimer dissociation and of the dimer-dimer affinity using kinetic means In the experiment

d e s c r i b e d a b o v e it is c l e a r

t h a t , o w i n g to t h e K m c h a n g e , t h e r a t e o f d i s s o c i a t i o n o f t h e t e t r a m e r to t h e d i m e r c a n b e f o l l o w e d by measuring the rate of decrease in the reaction velocity at a low pyruvate concentration. At a c o n c e n t r a t i o n o f 0.1 m M p y r u v a t e t h e d e c r e a s e i n r a t e is 2 - 3 - f o l d w h e n all t h e t e t r a m e r is c o n v e r t e d t o t h e d i m e r (see Fig. 4B), t h i s s h o u l d t h e r e f o r e b e

6

s u l t s f r o m s u c h a n e x p e r i m e n t a r e s h o w n i n Figs. 5A and B and reveal that the rate of tetramer d i s s o c i a t i o n is 1.5 • 10 - 2 s -1 a n d t h a t t h e d i m e r d i m e r d i s s o c i a t i o n c o n s t a n t is 0.11 /xM. F r o m t h e s e v a l u e s t h e r a t e o f a s s o c i a t i o n o f d i m e r s is 1.5 - 105 M -~ • s ] a v a l u e c l o s e to t h a t p r e v i o u s l y d e t e r m i n e d f o r w i l d - t y p e d i m e r s ( 2 . 5 . 1 0 5 M 1. s - ] [14]). T h e r a t e o f d i s s o c i a t i o n o f t h e w i l d - t y p e t e t r a m e r is g r e a t e r t h a n 1 s ] [14], t h e r e f o r e w e c o n c l u d e t h a t t h e g r e a t e r s t a b i l i t y o f t h e m u t a n t is d u e to this r e d u c t i o n i n t h e d i s s o c i a t i o n r a t e . T h e value obtained kinetically for the K d of the assemb l y p r o c e s s (0.11 f f M ) is i n a g r e e m e n t w i t h t h a t

4

t-"

2

amenable to measurement. Furthermore, the add i t i o n of e n z y m e f r o m s t o c k s o l u t i o n s o f l o w e r concentration, so that the dimer-tetramer equil i b r i u m is p o i s e d , s h o u l d y i e l d l o w e r i n i t i a l r a t e s . T h e s e i n i t i a l r a t e s a r e r e l a t e d to t h e i n i t i a l p r o p o r tion of tetramer and from these the K d for the a s s e m b l y e q u i l i b r i u m c a n b e c o n f i r m e d . T h e re-

obtained ffM).

A

from

polarization

measurements

(0.08

(5) Affinity of the enzyme-NADH interaction 0

o

A

4o

time (s) • "6

We have previously shown that the wild-type e n z y m e b i n d s N A D H w i t h a K d o f 1 . 5 - 2 . 0 ffM, i r r e s p e c t i v e o f t h e s t a t e o f s u b u n i t a s s e m b l y [13].

B

IJ

o! 0

,

i/

2.5

[d imerlo ,uM Fig. 5. Measurements of tetramer dissociation by kinetic means. Enzyme was added to cuvettes containing 0.1 mM pyruvate and 0.2 mM NADH to give a final concentration of 0.002 ffM dimer. However, the additions were made from stock enzyme solutions of differing concentrations and the reaction velocity was recorded, as a function of time, as the tetramer decayed to dimer on dilutuion. The decay in reaction velocity was exponential, and shown in (A) are example plots from three experiments; I , addition of 5 p.1 of 1 ~M enzyme (expressed as dimer); O, 50 ffl of 0.1 p.M enzyme; and (O), 100 tzl of 0.05 ~M enzyme. Vt is the reaction velocity at the given time and Vf the final velocity; both are measured in A A. min ~ × 10 4. Plot (B) shows the relationship between the stock concentration of enzyme (dimer) and the difference between the initial (Vi) and final (Vf) reaction velocities (measured in the same units as plot (A)). With a velocity difference between the dimer and the tetramer of 200 units, the enzyme concentration required for a measured difference of 100 units is 0.11 I~M (in dimers). This represents the value of the dimer-dimer dissociation constant.

1

1-Or

/

/

/

/

~o

2'o

~o

Fig. 6. The affinity of NADH binding to the mutant enzyme. A solution of 6 p~M NADH was titrated with the enzyme and the fluorescence polarization of the coenzyme was recorded (see Ref. 13). The plot shows the data transformed according to the method of Stinson and Holbrook [22], Lo is the total concentration of enzyme sites and ~ is the proportion of NADH molecules which are bound to the enzyme. The plot yields a K d of 4 #M and a sites concentration of 5.8 ~,M (i.e., one NADH binds to each enzyme subunit).

78

Fig. 6 shows the analysis of binding data (after the method of Stinson and Holbrook [21]) from a fluorescence polarization experiment in which N A D H was titrated with the mutant enzyme (for details of procedure see Ref. 13). The K a for the interaction is 4 / , M , it is not cooperative and there is one binding site per subunit. The experiment shows that the mutation has only a slight weakening effect on N A D H binding.

(6) The influence of quaternary' structure on the equilibrium binding of a substrate analogue Oxamate, an isoelectric and isosteric analogue of pyruvate, is a competitive inhibitor of the wildtype enzyme, its K~ matching the K m for pyruvate [13]. In addition, on binding at the active site, it quenches about 90% of the N A D H fluorescence.

Having these properties, it is a convenient reporter of changes in the substrate affinity of lactate dehydrogenase in equilibrium binding experiments. In Fig. 7 we present the results of two comparative experiments, one with wild-type and one with mutant lactate dehydrogenase, in which the apparent binding affinity of oxamate is determined over a wide range of enzyme concentrations. The plot shows that the mutant enzyme is converted to the tighter binding tetrameric form at much lower protein concentrations than is the wild-type enzyme; this merely confirms previous conclusions. It also shows that in the wild-type enzyme the K m improves 14-fold between the dimer and tetramer (2.2 0.15 mM), while in the mutant the improvement is only 7-fold (1.8 0.25 mM).

Discussion

X

x O

1

'\,

\

5> -

o

~

~ 10

JaM

~ 20

-0

J- "if' '

50

[dimer]o

Fig. 7. The relationship between o x a m a t e affinity e n z y m e c o n c e n t r a t i o n in the wild-type and m u t a n t lactate dehydrogenase. The plot c o m p a r e s the a p p a r e n t affinity for o x a m a t e of the two enzyme forms as the protein c o n c e n t r a t i o n is increased. The curves shown are for the wild-type e n z y m e ( O ) (taken from Ref. 13), and the m u t a n t (e). and all e x p e r i m e n t s were performed in the absence of F r u - l , 6 - P 2. The points at the lowest protein c o n c e n t r a t i o n s were d e t e r m i n e d by inhibition kinetics (as described previously [13]) and we a s s u m e that K, and K a are identical. The d i m e r c o n c e n t r a t i o n s at these low points are 0.003 ,uM for both enzymes. At higher protein c o n c e n t r a t i o n s the a p p a r e n t affinity' is d e t e r m i n e d by titrating the e n z y m e - N A D H complex with o x a m a t e and recording the q u e n c h in the coenzyme fluorescence, as explained in Ref. 13. For the points at which the d i m e r - t e t r a m e r e q u i l i b r i u m is poised, the K a (app) value is taken as the c o n c e n t r a t i o n of o x a m a t e required to saturate half the enzyme sites. This is d o n e because of the slight d e p a r t u r e from hyperbolic b i n d i n g at these protein concentrations; there is a small and predictable cooperativity owing to the k n o w n link between o x a m a t e b i n d i n g and subunit association.

Arg-173 is a residue which is conserved in all 15 of the known N A D H - d e p e n d e n t L-lactate dehydrogenase sequences; it is, therefore, surprising that its replacement by glutamine has not altered the properties of the enzyme more drastically. For instance, it has had little effect on the binding of N A D H and on the rate of pyruvate turnover, and in the unactivated dimeric form of the enzyme it has changed neither the K m for pyruvate nor the K d for its competitor, oxamate. In contrast, the mutation has markedly influenced the process of subunit assembly and the allosteric activation by Fru-l,6-P 2. In the mutant enzyme the tetramer is more stable than in the wild-type by a factor of 400, an extremely large positive effect for a single amino acid substitution (an increase of 3.6 kcal (14.5 k J) in the d i m e r - d i m e r binding energy). However, the symmetry relationship of the subunits in the tetramer (see Fig. 8) is such that two pairs of residue 173 face each other across the subunit interface on the P-axis of the protein. At least in part, we attribute the greater stability of the mutated tetrameric form to the loss of charge repulsion between R-axis dimers [13,14] as they assemble to bring the P- and R-axis interfaces together. Examination of the projected structure of the tetramer (see legend to Fig. 8) shows a 9-12 ,~ separation of opposing positive charges (in a medium with a dielectric

79

constant of 15 this arrangement

~~-207

+ FBP + tight

STABLE

-cl axis

MUTANT + FBP + weak

of the charge-repulsion hyFig. 8. Schematic representation pothesis. Shown in (A) is a view of the tetrameric wild-type lactate dehydrogenase molecule looking down the P-axis (a box of dimensions, in A,, 14x 14x 20 (P, Q, R) centred on 20,0, 0) and revealing the spatial relationship of residues across the subunit interface on this axis. The structure shown in generated by the PLUTO plotting routine from a model obtained by building the B. stearorhermophilus sequence into the coordinates of the pig H, enzyme s-lac-NAD structure [18] taken from the Brookhaven Protein Data Bank files. The atoms of residues belonging to the two different subunits are marked as open circles on one filled on the other. the two Arg-173 residues are made bold. The view clearly shows that the pair of Arg residues are close together (the guanidinium groups are 9-12 A apart) when the tetramer is assembled. Diagram (B) shows, schematically, the charge-repulsion argument which explains the relationship between Fru-1,6-P, binding and tetramer stability in the wild-type and mutant lactate dehydrogenases (see Discussion). The negative charge on Fru-1,6-P, which is usually paired with Arg-173 is represented by the symbol 8, and it is only this charge we are considering in the argument. Repulsive charge-charge interactions are denoted by the symbol (a------D). The P-axis related sites are close (around 10 A apart) but those across the Q-axis and on opposing dimers (i.e.. diagonally related on the picture) are separated by over 40 A.

of charge would render that of the mutant tetramer more stable by the observed factor). This result also supports our previous suggestion [13,14] that the tetramer is formed by dimers, in which the Q-axis interface is maintained, associating to bring the P- and R-axis interfaces together. The above observation can be used to explain why Fru-1,6-P, promotes tetramer formation in the wild-type enzyme. In this case Fru-1,6-Pz has previously been shown to bind to the dimeric form with a K, of 3 mM [14], but to assembled tetramer with aK, of 6-8 PM [13]. If the charge repulsion between the two pairs of Arg-173 residue across the subunit interface on the P-axis is responsible for the relative instability of the wildtype tetramer, then the binding of a ligand which overcomes the charge repulsion at these sites should be tighter to the tetramer than to the dimer (see Fig. 8(B)). Furthermore, of the four potential ligand sites in the tetramer, it is only occupation of the first two (one of each of the opposing pairs across the P-axis interface) which relieves the charge repulsion. Hence, these two Fru-1,6-P, molecules utilize not only the energy of an electrostatic interaction, but also that of relieving the unfavourable site-site repulsion in the tetramer. This mechanism helps to explain why we can detect, experimentally, only two bound Fru-1,6-P, molecules in the wild-type tetrameric protein [12,13] (see Fig. 8B). By this argument, further Fru-1,6-P, binding at the two remaining sites will be weak because it adds nothing to the stability of the tetramer. (In addition, it can be demonstrated that very high concentrations of Fru-1,6-P, cause the dissociation of the wild-type tetramer, suggesting that occupation of all four sites would lead to unfavourable Fru-1,6-P,-Fru-1,6-P, contacts (Clarke, A.R. unpublished results).) Consistent with this line of argument is the observation that, in the mutant tetramer, all concentrations of Fru-1,6-P, force dissociation to dimers. This can be explained by the loss of the positive charge in the Fru-1,6-P,-binding pocket and the consequent lack of charge repulsion. In these circumstances we suggest that, because there is no role for Fru-1,6-P, in relieving the repulsion between Arg-173 residues, there is no tight and stabilizing binding of a pair of Fru-1,6-P, mole-

80 cules to the tetramer. Moreover, we propose that occupation of all four sites generates repulsion between un-neutralized negative charges on Fru1,6-P2 molecules facing each other across the Paxis, forcing the tetramer to dissociate to dimers (see Fig. (8)). The results also show that this m u t a t i o n severely impairs c o m m u n i c a t i o n between the regulator site a n d the active site. This is best illustrated by the fact that when F r u - l , 6 - P2 binds to the dimeric form of the m u t a n t enzyme it has very little effect on its Kr,~ for pyruvate, and in fact acts as a mild inhibitor, whereas in the wild-type enzyme it is a strong activator reducting the K m at least 7-fold. This is the first strong evidence that Arg-173 is essential for the efficient transmission of the regulator signal to the active site residues, a role which we have only been able to speculate u p o n previously [13,14]. It is interesting to note that, although the residue is necessary for F r u - l , 6 - P2 regulation, m e a s u r e m e n t s of its b i n d i n g c o n s t a n t to the wild-type a n d m u t a n t dimers (3 m M and 8 raM, respectively) d e m o n s t r a t e that the substitution of Arg for G l n has decreased its b i n d i n g energy by only 0.6 kJ. This can be explained either by the formation of a hydrogen b o n d between the glutamine residue and a phosphate group of Fru1 , 6 - ~ , so that the whole of the residue 173 interaction energy is not lost, a n d / o r by F r u - l , 6 - P2b i n d i n g in the wild-type site being weakened by the ' p u l l i n g ' of an energetically u n f a v o u r a b l e protein structural r e a r r a n g e m e n t (we know that such a process must occur to transmit the b i n d i n g signal to the active site). There is a n o t h e r form of allosteric c o m m u n i c a tion in the protein, between the active site a n d the d i m e r - d i m e r s u b s u n i t interface i.e., the b i n d i n g of substrates is tight in the tetramer where these interfaces are in contact. The m u t a t i o n has a small effect upon this transfer of i n f o r m a t i o n , the wildtype showing an i m p r o v e m e n t in the K d for oxamate of 14-fold, a n d for the m u t a n t of 7-fold. It appears, therefore, that Arg-173 is quite specifically required to c o m m u n i c a t e F r u - l , 6 - P2 binding, but is less i m p o r t a n t in t r a n s m i t t i n g i n f o r m a t i o n between the p r o t e i n - p r o t e i n interface a n d the active site.

Acknowledgements We thank Professor M. Buehner for discussing with us his L. casei lactate dehydrogenase crystallographic structure and the S.E.R.C. for providing a project grant.

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