A kinetic study of ternary complexes in the mechanism of action of liver alcohol dehydrogenase

ARCHIVES

OF

BIOCHEMISTRY

A Kinetic

Study

AND

of Ternary of Liver JOSEPH

Nobel

Medical

116,

BIOPHYSICS

Complexes Alcohol

D. SHORE

Institute,

255-260

Department Received

(1966)

in the

Mechanism

of Action

Dehydrogenase’ AND

HUGO

THEORELL

of Biochemistr~y, May

Stockholm,

Sweden

18, 1966

It is shown that the ternary complex dissociation constants KER, sldehyde and Kxo, aloohol can be determined under certain assumptions from the maximum reaction velocity with and without added product. The results obtained with ethanol-acetaldehyde were in the same range of magnitude as those obtained by Theorell and Yonetani in 1963 by equilibrium measurement,s. The results with higher aliphatic alcohols clearly indicated the importance of a lipophilic binding site for the substrate.

Although the binary-complex ordered mechanism proposed in 1951 by Theorell and Chance (1) for liver alcohol dehydrogenase (LADH)z action provided an accurate description of the steady-state kinet’ics (2, 3), it did not exclude the cxistence of t,ernary complexes which dissociate and interconvert rapidly enough to have no effect on maximal velocity. The probable existence of ternary complexes has been mentioned repeatedly by Theorell and coworkers (2, 3) and was inferred from the production of ternary enzyme-coenzymeinhibitor complexes using substrate competitive inhibit,ors (4). Direct evidence for the existence of ternary complexes was provided by Theorell and Yonetani (5), who used spectrophotometric methods. Wratten and Cleland provided kinetic evidence for the existence of ternary complexes using product (6) and alternate product (7) inhibition. Consequently, it now seems fairly well established that enzyme-coenzyme-substrate complexes do exist but are difficult to discern with ordinary steady

state kinetics due to their rapid rates of interconversion and dissociation. In 1958, Alberty (8) suggested that in the presence of added product, the maximum velocity of an enzyme reaction such as t>hat of LADH would be decreased if ternary complexes existed but would be unaffected if only binary complexes existed. This theoretical basis was also used by Wratten and Cleland (6) in their kinetic demonstration of ternary complexes in the LADH mechanism. In the present paper, the change in maximum velocity due to the presence of product is used to calculate dissociation constants for ternary complexes in the LADH mechanism for various aliphatic alcohols. MATERIALS

AND

METHODS

Crystalline LADH was prepared from horse liver by the method of Dalziel (9), and the normality of the enzyme solution was determined as described by Dalziel (10). The calculation of normality was based on an equivalent weight of 42,000 and a specific extinction of 0.42 at 280 rnp for pure enzyme. All alcohols and aldehydes were freshly distilled before use. NAD+ and NADH were obtained from Sigma Chemical Company, and the NAD+ was purified by the method of Dalziel (11). Initial rate measurements were made with a recording fluorometer, previously described (la),

1 Dedicated to Luis F. Leloir on the occasion of his sixtieth birthday. 2 Abbreviations used: LADH, horse liver alcohol dehydrogenase; 0 or NADf, oxidized nicotinamide adenine dinucleotide; R or NADH, reduced nicotinamide adenine dinucleotide. 255

256

SHORE

AND

THEORELI,

In the general case the rate of breakdown of the ternary complex may influence the maximum xrelocity, and

which was modified by the addition of an 0lt)ronix U-524 power supply to the photomultiplier, which provided greatly enhanced sensitivit,y. ill1 kinet,ics were run in 0.1 p phosphate buffer at pH 7. The solutions used were filtered to remove as much dust as possible since this can be t,roublesome at high sensitivities. The stock enzyme solut,ions used varied from 0.5 to 3.0 ~LN, and 5-20 ~1 was added from the tip of a st,irring rod to initiate the reaction. The curves were extrapolated to zero time, and the slopes of the initial velocity were calculated. All kinetics were made in duplicate, and + and +’ values were calculated by the method elaborated by Dalziel (13).

According to the theory of Albcrty (S) the maximum velocity will be lowered by the presence of product in initial rate studies, since the product will tend to decrease the dissociation of t#ernary complex. Thus, in the presence of added product, the maximunl velocity t’erms will be :

RATIONALE

LADH conforms (1) to the general relationship proposed by Dalziel (13) for enzyme-coenzyme-substrate reaction mechanisms : 41 42 +(j = ” + [coenzyme] + [subst,rate] ’

[coenzymz;subst,rate]

’

In the cast of LADH, the coenzymc is NllDH and the substrate is aldehyde in the forward direction, while the cocnzyme is NAD+ and the substrate is alrohol in the reverse direction. Since the mechanism is symmetrical (3) the same relationship holds for the reverse reaction, using 4’ values. It can be seen that’ +. represents the reciprocal of the maximum velocity with NADH + aldehyde, and, similarly, 40’ represents the reciprocal of the maximum velocity with iYAD+ + alcohol. The meaning of each 4 value, in terms of rate const#ants, depends on the mechanism. From a kinetic point of view, the LADH mechanism is ordered (3, 6), and the presence of at least one ternary complex has been established (5, 6). It is therefore possible to write the mechanism of action in the following manner: E+R+ER

1

ER + aldehyde +

2

EXI’

+

3

EO

+ alcohol + Hf E+O

Since previous kinetic investigations (2, 15) indicated that LADH fits t,hc TheorellChance binary complex mechanism, it is reasonable to assume that the rates of dissociation of substrate produc*t:: from the ternary complex are very much greater than the rates of dissociation of cnzyme-coenzyme complexes. Therefore, X.a>> k4 and k-2 >> k-1, and the maximum velocity terms in the presence of product become ~ = l- + k-a[alcoholl 0

k3X.4

k.4

kz [alde hyde] k-z k-1 It can be seen from these relationships that by plotting 40 or $0’ against product concentration, and dividing the slopes by the intercepts, one can obtain k_.3/k3 and k,/k-, which are l/KEo,

alcohal

and

~/KBR,

aIdehyde.

The reciprocals of these values would thus provide the dissociation constants of the ternary complex in both directions. In the actual experiments, the intercepts of a curve of l/substrate vs. e/v at constant coenzyme concentration gave $0 + (+/[R]) in the forward direction and 40’ + (4,‘/[0]) for the reverse direction. The reverse reaction was easier to work with, since it was

LIVER

BLCOHOL

DEHYDROGENASE

possible to use high enough KAD+ concentration so that the +I’/[01 term was negligible. The effect of aldehyde concentration on ($0’ was thus readily obtained. In the forward reaction, it was not possible to use very high NADH concentrations due to fluorescence quenching. It was therefore necessary t,o determine 41 by separate experiments, and substract 41/[R] from t’he interceptjs obtained. Since the int,ercept in the presctlcc of alcohol is

~,.+

40

k---3 [aldd

WI

357

I

0.35.

7 a30-

I)____ 0:

+ &

ka

KINETICS

.

0.25. ’

and the intercept in the absence of alcohol is 40 + (:&/[R]), the following relationship will exist, : Intercept intercept

in the presence of alcoholwithout

alcohol =

40

0.15, 0

L3 [alcohol] k3

25

50

’

75

(Acelaldehyde)

IW

NM

2. The effect of acetaldehyde on $0’. NAD+ concent,ration, 656

concentra-

FIG.

tion

OI

,

1.0 1(EtOH)

20

3n

7

mM

FIG. 1. The effect of acetaldehyde on the LADH reaction wj th ethanol as variable substrate. NAD+ concentration, 656 PM. Acet,aldehyde concentrations: a, 0; 0, 44 pM; 0, 66 pM; 0, 110 MM.

5

10 I (Acetaldehyde)

125

PM.

15

20

mM

FIG. 3. The effect of ethanol on the LADH reaction with acetaldehyde as variable substrate. NADH concentration, 11.62 MM. Ethanol COW centrations: A, 0; 0, 6400 pM.

XIS

SHORE

AKD

THEORELL

and therefore

k-3 -=xl3

intercept difference [alcohol]$o

1 &O,nlrohol

It was possible to check the validity of our assumptions by determining & and +2’ experimentally since our theory rcquircs that

O5

/’

,//’

,,”

’/,”.

. OL

I

////

/

RESULTS

03

It has already been established by Wratten and Cleland (7) that the C2-C, primary alcohols and their corresponding aldehydes follow an ordered mechanism in which dissociation of coenzyme is the last step. Our data have reaffirmed this since 40, 4: and $1, 41’ were the same for the alcohol-aldehyde pairs used in this study, within the limits of exnerimental error (A 10 %). It has thus been possible t’o determine the KER, a&h,& and KEO, alcoholvalues for the ethanol-acet’aldehyde, propanolpropionaldehyde, butanol - but>yraldehyde, and pentanol-valeraldehyde pairs.

/ J

o2 25 i Propionoidehyde)

FIG.

5. The effect on ~$0’. NADf

centration

50

75

pM

of

propionaldehyde concentration,

co,,656 PM.

r 0.25

A

020

015 20. Q ” 010

0 05

C

--IO 1 (Ropmaldehyde)

L

0'5 i&j-r

10

15

mM

FIG. 4. The effect of propionaldehyde on t,he LADH reaction with propanol as variable substrate. NAD+ concentration, 656 pM. Propionaldehyde concentrations: A, 0; 0, 51 PM; 0, 85 MM.

20

33 mM

FIG. 6. The effect of propanol on the LADH reaction with propionaldehyde as variable substrate. NADH concentration, 11.62 PM. Propanol concentrations: 0, 0; A, 6600 PM.

The inhibition of the LADH reaction by acetaldehyde, with ethanol as substrate, is shown in Fig. 1, and the effect of acetaldehyde concentration on the 40’ value is

LIVER TABLE KINETIC

COEFFICIENTS

ALCOHOL

DEHYDROGENASE

I AND

DISSOCIATION

CONSTAHTS

Sllbstrate

Q,n’

I

$52

I..

C’z 98 co 41 c 4 ~ 8.3 c, 2.1

‘4.7 0.48 -

180 83 39 13

-

4550 2420 757 433

plot#ted in Fig. 2. Dividing the slope of Fig. 2 by the intercept yields the reciprocals of K ER, Rects~dchyde.Figure 3 indicates the inhibit#ion of the reaction by ethanol wit’h acetaldehyde as substrate. It was possible to calculate the reciprocal of KEO, alcohol by using the formula derived in this paper, wit’h $0 equal to 0.01. Figures 4, 5, and 6 show t,he same type of dat,a, obtained with propanol and propionaldehyde substrates. Similar st,udies were also performed with butanol-butyraldehyde and pen-tanol-valeraldehycle subst’rates, but these diagrams were not included. Table I summarizes the data obtained from various substrate pairs. The 4Z’ and 42 values listed were obtained by independent experiments in which the method of Dalziel -was used (13). The final column shows the +2/+2’ ratio, which, according to our assumptions, should be equal t,o Km.

ald~hyde/%o.

alcohol.

This correlation is seen to obtain for CZ and C, subskates, the only ones for which 4Z values were determined. DISCUSSION

The only data available in past literature on ternary complex dissociation const’ants are those of Theorell and Yonetani (5) for ethanol .and acetaldehyde. Their values for KER, a&a&hhy& ranged from 84 to 295 PM, and for K,,. alcohalwere between 1120 and 10,000 PM. Consequently, our results seem to be within the same general range and order of magnitude. This correlation seems particularly interesting since their data were ob-

2.59

KINETICS

tained by direct observat,ion of equilibrium mixtures. The importance of lipophilic binding of substrates t,o enzyme-coenzyme complexes had been previously inferred from the work of Theorell and Bonrlichscn (16) with K, values for several alcohols, Dalziel (14), who found very large differences in +? and 42’ values for CZ and Cd substrnt#es, and the work of Wirier rind Thcorell (4) 011 the dissociation constants of t,ernary complexcs of fatty acids and fatt#y acid amides with enzyme-cocnzyme complexes. S’ i lIl(‘C fatty acids were compet’itive with alcohol and t,he amides mere competitive w&h aldehycle, the remarkably strong effects on ~TEo.Reid and KER, amide caused by increasing chain length indicated the significance of lipophilic interactions. In agreement herewit,h the clissociation constants of the coenzymcs from these ternary complexes are not influenced by the C-chain length of the inhibitors. The results obtained in t,his paper tend to subst’ant’iatc the importance of these interactions, since t,here was a more than tenfold difference in the dissociation constants for the CZ and Cg substrates. The correlations between K

ER, aldehydc/&m

nlcohol

and &/& for CZ and Ck substrates may be interpreted as support for tfhe I-alidity of the theoretical basis of this work. If the term l/k3 had a significant effect on +. , and l/Lz was important in the 40 term, the ratio Ii ER , ald~hyde/&O, alcohol would deviate considerably from the +J+Z’ rat’io. Another interesting relationship is the relatively constant, values for the ratio of dissociation constants for all t#he subskate pairs tested. This provides further evidence for the great importance of the lipophilic interaction in the binding of both alcohols and alclehydcs to t,he enzyme-coenzyme complex. The ternary complex dissociation COIIstants calculated in this paper are all based 011 t#he assumption of only one ternary cornplex in the mechanism. It is probable, however, that there arc at least t,wo ternary complexes. In that case the following st,ep would have to be included:

260

SHORE

.4ND

ER, nltlchyde -+ EO, alcohol + Hf. This would result in the dissociation COIN&ants listed in Table I really bring equal t,o Ii H, n~i~e~,ydc: [l + (k/k’)] and KKO, a~co& 11 + /;‘,/I<]. The true dissociat,ion caonstnnts would thus be larger than the values given. The +J+2’ rat#io would be equal to

01’

TIIFNIIELL

EO,alcohol, which is the most, important step in ihe reaction mechanism.

This work wits supportedby a postdoctoral fellowship to J. D. Shore from the Muscular Dystrophy Association of Bmerica, Inc., and grants from Institutet far Maltdrycksforskning. The technical assistance of Mrs. C. Popper is gratefully acknowledged.

L3,aldehydc k’ K E",alcohol

k '

KEFEIXXCES L

1. TFIEORELL, II., .IND CHMVCE, B., Ida

Chem. 5, 1127 (1951). THEOREM,, H., ASU RIC'KINLEI--RIcHEE, J. S., Scfe Chem. Stand. 15, 1797 (1961). SUND, H., AM TIIEORELL, H., in “The Enzymes” (P. D. Bayer, 11. Lardy, and K. Myrb$ck, eds.), 2nd edition, Yol. 7, p. 25. Academic Press, New York (1963). WINER, 8., AXI) THEORELL, II., Bela Chem. &nnc1. 14, 1729 (1960,. THEOHELL, II., .ZXD YONET.ISI, T., drch. Uiothem. Biophys. Szrppl. 1, 209 (1962j. WRITTEN, C. C., AND CLEL.ZND, W. W., Biochemistry 2, 935 (19G3). WMTTEN, C. C., AN) CLELLNU, W. W., Bioche,n&ry 4, 2442 (19ti5). ALBERI>I-, R. A., J. A1?n. C’he,,a. Sot. 80, 1777 (1958). DALZIEL, K., dctn. Chem. Scunrl. 12,459 (1958). D.ILZIEI,, K., Acta. Chew. Stand. 11, 397 (1957). DALZIEL, K., J. Bid. Chem. 238, 1538 (1963). A. P., ilcta. THEORELL, H., AXU NYG.\.IRD, Chem. Xcnnd. 8, 877 (1954). D~LZIEL, K., Bela Chem. Scnnrl. 11, 1706 (1957). DALZIEL, K., Biochem. J. 84, 244 (19tX). DI\~zl~~, K., Biol. Chem. 238, 2850 (1903). THEORELL, H., END BONMCHSEN, IX., dcta Chem. &and. 6,1105 (1951). Scnnd.

whkh is the expcctecl relationship for a mechanism with two ternary complexes (I:<). Unfortunntcly, in the absenceof a method for measuring t(he two ternary complexes directly, it is not possible to determine the V/k ratio. The interconversion of the tcrnnry complexes is probably even more complicnt’ed than a simple one-step reacation since the proton and hydride ions are likely to be removed from or added to the substrate in separate steps. i2lthough the relationship of rhnin length to substrate affinity is important, the most significnntJ aspect, of this work is that it provides an indication of the order of magnitjude of the dissociat’ion constant,s of the ternary complexes. From these dat)a, plus the known dissociat,ion constants for the binary complexes (3), it should he possible to calculate the conrcntrat,ions of substrates and products necessary to produce substantial concentrations of t’ernsry complexes. It might then be possible to obtain more informat)ion about t)he rate of interconversion between ER,aldehycle and

,

2. 3.

4. 5.

6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16.