Some kinetic studies on aldehyde oxidase

BIOCHIMICA ET BIOPHYSICA ACTA

135

BBA 13658

S O M E K I N E T I C S T U D I E S ON A L D E H Y D E

OXIDASE

GRAHAM PALMER*

Department of Biochemistry, University of Shejfield, She~eld (Great Britain) (Received January I8th, 1962)

SUMMARY

The kinetics of the reduction of 2,6-dichlorophenolindophenol and cytochrome c by pig-liver aldehyde oxidase have been examined. The results suggest that the reaction mechanism involves ternary complex formation with independent binding of each substrate, With ferricyanide as electron acceptor a different reaction mechanism seems likely. The inhibition of the reduction of cytochrome c by I,Io-phenanthroline has been investigated and shown to be partially competitive in nature. The inhibition produced b y p-chloromercuribenzoate is truly competitive with acetaldehyde and suggests the aldehyde substrate is bound to the enzyme as a thiohemiacetal. Experiments on arsenite inhibition are also reported. INTRODUCTION

Oxidizing enzymes catalyze two substrate reactions of the type S 1 + S~ ~ S~ + S 4

for which theoretical treatments analogous to those of MICHAELIS AND MENTEN1 and BRIG(;S AND HALDANE2 for single substrate reactions have recently been developed as a basis for experimental study3, 4. For those flavoproteins so far examined, 2 quite different reaction mechanisms have been found. The first, which has been observed with D P N H cytochrome c reductasO, m a y be called the independent binding mechanism. Here the enzyme forms a ternary complex with both of its substrates the combination of the enzyme with S 1 having no effect on its affinity for S~ and vice versa. This mechanism requires that the slope of the double reciprocal plot -i VerSUS v ~S1] is a function of [S~, while the negative intercept on the abscissa is independent of [S~] (See ref. 5 for further details). Abbreviations: DCIP, 2,6-dichlorophenolindophenol; BSA, bovine serum albumin; PCMB, p -chloromereuribenzoate. * Present address: Institute for Enzyme Research, Madison, Wise. (U.S.A.).

Biochim. Biophys. ,4,ta, 64 (I962) 135 148

136

c,. PALMER

The second mechanism to which most flavoproteins studied so far seem to conform, involves successive reduction and oxidation of the enzyme itself, b y reaction i

with each substrate in turn, The slope of the plot v versus ~

i

is independent of IS2J

and consequently a series of parallel lines would be obtained if the experiment is performed at several concentrations of S~. For brevity, we shall call this the reduced enzyme mechanism. It has been observed with lipoyl dehydrogenase 6, D-amino acid oxidase ~ and glutathione reductase 8. I t was considered of interest to see to which, if either, of these two mechanisms aldehyde oxidase would conform and the results of these experiments are reported here. The nature of the inhibition of enzyme activity b y orthophenanthroline, PCMB and arsenite has been investigated and the results are also reported in this paper. The relation of these results to the mechanism of action of this enzyme is discussed. MATERIALS AND METHODS The preparation of aldehyde oxidase and the assay conditions have already been described 9. Unless stated otherwise, the enzyme employed had a specific activity of 2.4 (DCIP assay). The source of most of the reagents has already been reported 9.

1.4

1.2

1.0

0.8

0.6

-2

200 1 [Acetoldehyde] (M-l)

400

Fig. I. Plot of reciprocal initial velocity versus reciprocal acetaldehyde concentration at 4 concentrations of DCIP. i k - - A , 67/~M; ~ - - I D , 33/~M; I - - I , I7 # M ; 0 - - 0 , IO/2M. Biochim. Biophys. Acta, 64 (I962) I35-I48

137

KINETIC STUDIES ON ALDEHYDE OXIDASE

PCMB was a gift from Professor Q. H. GIBSON. i,io-Phenanthroline was obtained from the Sigma Chemical Company (St. Louis, Mo.) and arsenite from the British Drug Houses. RESULTS

When acetaldehyde was varied at several concentrations of DCIP a series of straight lines were obtained which converged to a common point on the abscissa. The results are shown in Fig. I. A similar result was obtained when, in a separate experiment, DCIP was varied at 2 concentrations of acetaldehyde (Fig. 2). From considerations

1.6-1.4--

14

1.0

O.e--

,

0.4

1

0.06

0.12

[oc,P] (,~M-') Fig. 2. Plot of reciprocal initial velocity v e r s u s reciprocal DCIP concentration at 2 concentrations of acetaldehyde. O - ~ O , 6 m M ; O - - - O , 33 m M .

7

6

4 3

I

400

BOO

1200

1600

I

2000

1

[Acetaldehyde](M-1J Fig. 3. Plot of reciprocal initial velocity versus reciprocal acetaldehyde concentration at 4 conc e n t r a t i o n s o f c y t o c h r o m e c. I - - I , I5.4 ~3¢; 0 - - 0 , lO. 3 ffM; [ ~ - - [ ~ , 5.I # M ; 0 - - - t l , 3.1 ffM. Biochim. Biophys. Acta, 64 (1962) I 3 5 - t 4 8

138

G. PALMER

already detailed in the introduction, these results exclude the reduced enzyme mechanism and are consistent with the alternative mechanism of ternary complex formation with independent binding of each substrate. When these experiments were extended to cytochrome c a similar graphical

I

I

I

1

I

I

0.04 0.08 0:12 0.16 0.20

1

ECytochromec]

I

(HM.1)

Fig. 4- Plot of reciprocal initial velocity v e r s u s reciprocal Cytochrome 6 concentration at 2 concentrations of acetaldehyde. 0 - - 0 , 2.5 mM; O - - O , I2.5 mM.

picture was obtained (Figs. 3 and 4), indicating that this mechanism is common to at least 2 of the electron acceptors employed. Such a simple interpretation was not possible for the kinetic studies made with the acetaldehyde-ferricyanide system because of (a) the pronounced curvature of the double reciprocal plot (Fig. 5) and (b) the absence of any variation of enzyme activity on varying the ferricyanide concentration. However, preliminary measurements with crotonaldehyde may be inter-

1.0

13 °-eF

o4k 0.2

I

I

40 I

I

I

80 (M-I)

l

I

120

EAcetaldehyde]

Fig. 5- Plot of reciprocal initial velocity v e r s u s reciprocal acetaldehyde concentration at one concentration of ferricyanide (o.33 mM).

Biochim. Biophys. Acta, 64 (1962) 135-148

139

KINETIC STUDIES ON ALDEHYDE OXIDASE

preted as suggesting that the independent binding mechanism is not operating when ferrieyanide is the acceptor. This mechanism requires that Kaldehyde is independent of the acceptor used. Thus with DCIP and cytochrome c, Kalaehydeis 5.2 mM (Fig. I) and 5.0 m M (Fig. 3) and is in excellent agreement; similarly, with these acceptors, Kcroto.~adehyd~is X.O and 1.2 mM. With ferricyanide however a value of 6. 7 m M was obtained for Kcrotonaldehyde. This is substantially different from that obtained with DCIP and cytochrome c as acceptors and suggests that a different mechanism is

o8i ~

0.6

0.2

I

0.02

I

1 0,04.

I

0.06

I

I

0.08

0.10

[Dc~p] (#M-,) Fig. 6. Plot of reciprocal a p p a r e n t m a x i m u m velocity at infinite aldehyde concentration (Fig. t) versus reciprocal DCIP concentration.

operating, although it must be pointed out that the independent binding mechanism has not been confirmed with crotonaldehyde as primary substrate. I

KDcIp has been determined directly from the primary plot of ; I

versus ~

as 7.o #M. From the secondary plot 5 of Fig. I Vacetalaehyaev e r s u s ~

I

i

(Fig. 2)

a straight line

~ 1.0

~0.~ ,~0.6 0.4 0.2--

I

0,1

I

I

1 0.2 0.3 [Cytochrome c] ()JMJ)

Fig. 7. Plot of reciprocal a p p a r e n t m a x i m u m velocity at infinite aldehyde concentration (Fig. 3) versus reciprocal cytochrome c concentration. Biochim. Biophys. Acta, 64 (I962) 135-148

14 °

G. PALMER

was obtained (Fig. 6) and KDCIP evaluated as 6.2 ffM, a value which agrees well with the direct experiment. Kcytochromec was determined directly as 6 # M (Fig. 4) while a value of 5.2 # M was obtained from the secondary plot (Fig. 7) of Fig. 3.

Kinetic analysis of the inhibition of cytochrome c reduction by orthophenanthroline One role for the metal for which the available evidence is not unreasonable, is that this component functions as the binding site for one-electron accepters. The published figures demonstrating competitive inhibition between accepter and metalchelating agents, e.g. citrate, pyrophosphate, are impressive n, and it appeared important that these observations be extended to aldehyde oxidase which is thought I

I

to contain molybdenum. A series of double reciprocal plots v v e r s u s cytochrome c were obtained in the absence, and in the presence of several concentrations of the inhibitor, I,Io-phenanthroline. The straight lines so obtained converge and intercept at

I

-Vmax (Fig. 8), i.e. Vmax is independent of the inhibitor concentration while the apparent

2°I 16

I~ a t -

e.1

0.2

Fig. 8. Plot of reciprocal initial velocity versus reciprocal cytochrome e concentration in the absence a n d presence of several concentrations of orthophenanthroline. O - - O , no orthophenanthroline; A - - A , 7.5 ffM orthophenanthroline ; O - - O, i5 ° ffM orthophenanthroline.

Km of the enzyme for cytochrome c increased with increasing inhibitor concentration. It would thus seem that in this system I,Io-phenanthroline inhibits the enzyme in an essentially competitive manner. Consequently it should be possible to calculate Ki from the equation: K~

I . Ks Kp K8 -

(i)

-

Biochim. Biophys. Acta, 64 (1962) I35-I48

KINETIC STUDIES ON ALDEHYDE OXIDASE

141

where I is the concentration of I,IO-phenanthroline, Ks the Michaelis constant of enzyme for cytochrome c and K~ the apparent Michaelis constant for enzyme for cytochrome c in the presence of the inhibitor. The results of such a calculation at 4 concentrations of i,Io-phenanthroline are given in Table I. It is obvious that there is no agreement between the values of K, and it would thus appear that the inhibition is not genuinely competitive in nature. DixoN AND WEBB in their recent book on enzymes l~ have described a novel form of competitive inhibition which they called partial competitive inhibition. This was proposed to occur when the inhibitor is bound adiacent to the substrate binding site TABLE I Concentration of orthophenanthr oline

Kt (competitive) *

K , 1 (partially competitive) **

(,am)

(I~M)

(I~M)

620 15° 3° 7.5

200 51 17 5.0

1.9 5-4 3.2 2.0

" C a l c u l a t e d f r o m E q n . i. ** C a l c u l a t e d f r o m E q n . 6.

with a consequent effect on the affinity of the enzyme for the substrate. The breakdown of both E S and E I S (enzyme-inhibitor-substrate complex) occurs at the same rate. This situation can be depicted thus: Ks k E + S ~ES-+E +I

+ P

+I

~ K~

~ K~"

Ks" k E I + S ~- E I S -+ E I + P From

D I X O N AND W E B B 12

we have V

(2)

--

7~7|I [ - Kss I

I

/

where I is the concentration of inhibitor; S, the concentration of substrate reaction velocity and e the enzyme concentration; and Ks K( [(s" - - - Ki

v, the

(3)

I t follows that I Kp

Ki K x ( K i + I)

+

I. Kt K s K~" ( K i + I)

(4)

I

I

+Kt

(5)

K~

Ks'

When I >> K, I

Ks I Biochim. Biophys. Acta, 64 (1962) i 3 5 - 1 4 8

I42

C. PALMER

that is, the intercept of the plot ~

I

I

,

I

versus i is Ks's'" Furthermore [ I -- [•P] L7 ]

/

(6)

I

On plotting ~

I,Io-phenanthroline (Fig. 9) a non-linear relation was obtained which was extrapolated to a value of Ks' of 1.34" IO-4 M at infinite I,Io-phenanthroline concentration. Although the curve is not linear its disposition is such as to make this extrapolation reliable. Values of K, subsequently calculated from Eqn. 6 are given in Table I. An average value of 3.2" IO-6 M was obtained for K,. K i ' can be determined from Eqn. 5 and all 4 equilibrium constants are thus versus

13

11

-f~

g

I

I

0.04

I

I

I

t

0.08 0.12 1 [1.10 phenar/chroline] OjM'I)

I

0.14

Fig. 9. Plot of the reciprocal of Kp versus reciprocal orthophenanthroline concentration.

obtained. They are: K s = 3.3" IO-5 M (by experiment), Ks' = 1.34" lO -4 M (from Fig. 9), K, = 3 . 2 . I o - ~ M (from Eqn. 5), and K / ' = 1 . 2 7 " I o - 5 M (from Eqn. 3)The much better agreement obtained when Ki was calculated according to Eqn. 6 indicated that the inhibition of the enzyme by I,Io-phenanthroline was partially competitive in nature. This conclusion was confirmed by the simple test described by DIXON AND WEBB 12. The enzyme activity was measured under standard conditions in the presence of increasing amounts of the inhibitor; it was found that the activity was not completely abolished but decreased to a finite and measurable value when all the enzyme is present as E I (Fig. IO). I f competitive inhibition was in operation it would be expected that at sufficiently high concentrations of I,IOphenanthroline complete inhibition would be obtained.

Sulfhydryl reagents and aldehyde oxidase The inhibition of an enzyme b y a reagent known to react with sulfllydryl groups is conventionally interpreted as demonstrating that a flee sulthydryl group is required for the unimpaired activity of the enzyme. Although this conclusion will usually be correct, it is important to note that the more common sulthydryl reagents are not completely specific for - - S H groups and that other enzyme groups m a y be involved. A more clearcut, but not necessarily unambiguous result, m a y be obtained Biochim. Biophys. Acta, 64 (1962) 135-148

I43

KINETIC STUDIES ON ALDEHYDE OXIDASE

by using several reagents, each of which react with the sulthydryl group in a different way, e.g. mercaptide formation, alkylation and addition. MAHLER and his colleagues 13 reported the inhibition of aldehyde oxidase by PCMB, iodoacetate and 2_methyl_5,8_dihydroxy_i,4_naphthoquinone , reagents which perform the reactions just noted, and it would thus appear reasonable that aldehyde oxidase contains

O.lO O.OE u~

O.Oe 0

• L 0.04 --~ 0.0¢

I

I

2

I

I

4

I

I

6

I

I

s

[1.10-phenanthroline] (M×IO 4)

I

Fig. io. Effect o f o r t h o p h e n a n t h r o line on t h e r e d u c t i o n of c y t o c h r o m e c b y a l d e h y d e oxidase. T h e enz y m e a n d i n h i b i t o r were i n c u b a t e d t o g e t h e r a t o ° for IO rain a n d t h e activity determined thereafter un1 der s t a n d a r d a s s a y conditions. T h e I e n z y m e u s e d for t h e s e e x p e r i m e n t s 10 h a d a specific a c t i v i t y of 1.6 ( D C I P assay).

an essential sulfhydryl group. One possible role for this group is the binding site for the aldehyde substrate according to the reaction H

I

E-SH + RCHO m E--S--C--R

I

OH

i.e. the reversibie addition to the aldehyde carbonyl group with formation of a

thiohemiacetal. The sensitivity of the enzyme to PCMB was confirmed and some kinetic studies were made to elucidate the mode of action of the inhibitor. At first the results were complicated by an unsuspected reaction of the PCMB with the BSA present in the enzyme assay system (Table II). T A B L E II 4 o f f g of e n z y m e p r o t e i n a n d r e a g e n t (as indicated) i n c u b a t e d in t h e s p e c t r o p h o t o m e t e r cell for i rain a t p H 8.5 a n d 25 °. T h e r e a c t i o n w a s s t a r t e d b y t h e a d d i t i o n of D C I P a n d a c e t a l d e h y d e . Activity DGIP

Conditions Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme

+ + + + + +

EDTA EDTA PCMB PCMB PCMB PCMB

(units/rag protein)

(0.5 ffmole) + B S A (3.3 mffmoles) (o.io ffmole) + EDTA + BSA + BSA + EDTA

i .65 2.6 2.6 0.8 1.3 2.6 2.6

Biochim. Biophys. Acta, 64 (1962) 135-148

I44

g. PALME~

These results can be explained by assuming that the BSA present in the assay mixture is able to remove any PCMB bound to the enzyme and consequently reverse any inhibition which might have occurred. Significant inhibition was not obtained until 2.5 #moles of inhibitor were added to the assay system. Presumably at this level the BSA is saturated with PCMB and the excess inhibitor is then available to react with the enzyme. It is interesting that no such effect was obtained with EDTA, which is reported to complex PCMB (see ref. 14). In view of this reaction of BSA it was omitted from the assay system for these experiments. A kinetic analysis of the inhibition of PCMB (Fig. I I ) was made by studying the effect of acetaldehyde concentration on the extent of the inhibition and a graphi-

B

2£

/

1.(}

~1.2

0.8

0.4

I

200

I

I

400 600 1 (M-~) EAcetoldehyd~

Fig. I i. Plot of reciprocal initial velocity versus reciprocal acetaldehyde concentration in the presence of PCMB, using DCIP as electron acceptor. All additions directly to the spectrophotometer cell. Q - - O , no PCMB; A - - A , 12o ffM PCMB; I I - - It, 18o ffM PCMB; / k - - A , 240 ffM PCMB; O--- O, 54 °/~M PCMB.

cal picture typical of competitive inhibition was obtained. Calculation of Kt using Eqn. I yielded values of 0.55, 0.45, 0.40 and 0.75 m M at concentrations of PCMB equal to o.12, o.18, 0.24 and 0.54 mM. These results, in contradistinction to those obtained with orthophenanthroline agree satisfactorily and suggest that competitive inhibition is operating in this system. Calculation of K, assuming partial competitive inhibition using the method described earlier, produced values ranging from IO to 3 ffM. Furthermore it was possible to obtain complete inhibition of the enzyme with suitably high concentrations of inhibitor. Biochim. Biophys. Acta, 64 (1962) 135-148

KINETIC STUDIES ON ALDEHYDE OXIDASE

145

Similar experiments were also performed with arsenite, a reagent which reacts with dithiol groups (Fig. 12). Once more the pattern typical of competitive inhibition was obtained although the values for K,, assuming pure competitive inhibition, are not in satisfactory agreement. These values are 4.6, 2.7 and 1. 9 m M at concentrations of arsenite of I.I, 1.6 and 2.o m M respectively. Attempts to calculate Ki, assuming partial competitive inhibition as before, were not possible as I

I

a plot of 37~ against arsenit~ did not have a sensible slope.

1.8 -

o/

1.6~

"i 1.2

/

¢

0.8

0.4

I

I

I

200

400

600

1 (M4) [Acetaldehyde~

Fig. 12. Plot of reciprocal initial velocity versus reciprocal acetaldehyde concentration in the presence of arsenite using DCIP as electron aceeptor. All additions directly to the spectrophotorneter cell. 0 - - O , no arsenite; A - - A , I m M arsenite; B - - It, 1.6 m M arsenite; C)--(J, 2 m M arsenite.

It might thus be concluded that both PCMB and arsenite can combine reversibly with the enzyme sulfhydryl responsible for binding acetaldehyde, and that, in all likelihood, a second - - S H group is nearby on the enzyme surface. DISCUSSION

The results presented in this paper would appear to eliminate the reduced enzyme mechanism as the reaction pathway during the reduction of DCIP and cytochrome c. The results are consistent with the independent binding mechanism, which has been demonstrated previously with D P N H cytochrome c reductase 5. However, it should be pointed out that the graphical picture presented here might sometimes be found Bfochim. Biophys. Acta, 64 0962) i35-148

146

G. PALMER

with systems which actually follow other reaction mechanisms due to a fortuitous combination of rate constants, and a comprehensive study of the effect of p H on both the forward and reverse reaction is really required to eliminate the common alternatives. Unfortunately with this enzyme it has not been possible to study the reverse reaction at all. With the independent binding mechanism, the m a x i m u m velocity of the reaction can be qualitatively interpreted as the rate of the molecular rearrangement of the ternary complex E S t S ~ ~ ES~S~

An additional conclusion is obtained from the results with cytochrome c. The overall reaction m a y be written C H s C H O + H 2 0 + 2 c y t o c h r o m e c 8+ -_~ CH3COOH + 2 c y t o c h r o m e c 2+

I f both molecules of cytochrome c reacted simultaneously with the enzyme, then the double reciprocal plot of velocity against cytochrome c concentration would be expected to show a linear dependence

i o n [cytochromec]2

Actually the results ob-

i

tained show a good linear relation with cytochrome c (Fig. 3)- A similar result has been obtained b y FRIEDEN 5 studying D P N H cytochrome reductase who pointed out that if 2 cytochrome c molecules reacted in succession, and the reaction of the second molecule was very fast then kinetic results appropriate to a 2-substrate reaction would be obtained. I t has not been possible to arrive at any firm conclusions with regard to the mechanism with potassium ferricyanide as acceptor, but there are indications that it differs from the other acceptors employed. It is interesting to note that with those flavoproteins for which the natural substrates are used, viz. lipoyl dehydrogenase ~, glutathione reductase 8 and D-amino acid oxidase 7, the reduced enzyme mechanism has been found to operate. The 2 enzymes which conform to the independent binding system have been studied either with an acceptor of an obviously artificial nature, or with an acceptor of unknown physiological significance in relation to these enzymes. It m a y well be that when these latter enzymes are reinvestigated using their natural acceptors, they too will be found to follow the reduced enzyme mechanism. When ferricyanide and cytochrome c were employed as electron acceptors, marked deviation from linear double reciprocal plots were obtained at high concentrations of aldehydes. These deviations were of the form usually attributed to inhibition by excess substrate. The usual explanation of this inhibition is that a second molecule of the substrate combines with the enzyme-substrate complex to form an enzymically inactive complex. Although this is likely to be the true explanation of this phenomenon for enzymes catalyzing single substrate reactions, DALZIEL4 has shown that such deviations from linearity are inherent in the initial rate equation for reactions which follows the general 2-substrate mechanism, and that these considerations might also explain the apparent activation of some enzymes obtained at high substrate concentrations, e.g. yeast alcohol dehydrogenase 1~. Biochim. Biophys. Acta, 64 (1962) 135-148

KINETIC STUDIES ON ALDEHYDE

OXIDASE

I47

Consequently, when studying 2-substrate reactions, upward deviations of double reciprocal plots should not, in the absence of further evidence, be interpreted as demonstrating the binding of a second substrate molecule b y the enzyme-substrate complex (cf. ref. I6). I t is clear from the experimental section that the Michaelis constants for cytochrome c obtained at different times were completely different. The reason for these variations is not obvious although some possibilities spring to mind. The initial high values were obtained with earlier preparations of the enzyme which had been purified b y a modified method, and it is conceivable, that the differences in extraction of the enzyme produced some minor changes in its activity, or possibly resulted in the presence of an inhibitor in the enzyme preparations. A kinetic analysis of the inhibition produced by i,Io-phenanthroline demonstrated clearly that the metal does not function as the binding site for the tyrochrome c. However, the results suggest that both the molybdenum site and the cytochrome c binding site must be very close to one another on the surface of the protein. Alternatively the binding of the inhibitor at a distant site might induce conformational changes in the protein which result in the observed decrease in the affinity of the enzyme for its substrate. Although the experimental results produced a graphical picture typical of competitive inhibition the calculation of Ki at several inhibitor concentrations revealed that the conventional "true competitive inhibition" was not operative. More elaborate considerations were required to show that the alternative partial competitive inhibition was in force. These results illustrate clearly the necessity for more thorough investigation of the numerous claims of competitive inhibition which abound in the literature, claims which very often result from the inspection of graphical results obtained with one concentration of inhibitor. The simplest method of distinguishing between the 2 systems depends on the property of the latter type of inhibition of not producing complete inhibition, so that the apparent activity will decrease to a constant value at high inhibitor concentrations. I t has been possible to utilize the equations derived by DIXON AND WEBB lz, who were the first to consider this situation to show that not only Ks', the affinity of acetaldehyde for the enzyme inhibitor complex but also Ki and Ki' can be obt

I

tained. K s is obtained b y plotting K~p v e r s u s

I

orthophenanthroline and extrapolating

the line obtained to infinite inhibitor concentration. Although the equation for this relationship is not linear in form, it does reduce to a simple form when the concentration of the inhibitor is substantially greater than K,. Extensive studies have been made on the mechanism of action of aldehyde dehydrogenases b y 2 groups of workers. STOPPANI AND MILSTEIN 17-2° have, from an examination of several enzymes, presented evidence which indicates t h a t the pyridine nucleotide is bound to the enzyme b y means of an enzyme sulihydryl group as has been suggested for the "alcohol dehydrogenase"-type enzymes ~1. They found t h a t the appropriate pyridine nucleotide, but not the aldehyde, protected the enzymes studied from inhibition b y sulfiaydryl reagents. JAKOnY and coworkers x6 however, have arrived at the opposite conclusion. They found t h a t proteolytic digestion of suecinic semialdehyde dehydrogenase is greatly increased b y the addition of pyridine nucleotide to the native or PCMBBiochim. Biophys. Acta, 6 4 (~962) i 3 5 - t 4 8

148

G. PALMER

treated enzyme, although the latter did not possess any increased sensitivity to digestion. Furthermore, they showed that inhibition of the enzyme by arsenite is apparently competitive with respect to succinic semialdehyde. They concluded that whereas the aldehyde is bound as a thiohemiacetal, the pyridine nucleotide is not linked to the enzyme via a sulfhydryl group. The results obtained with aldehyde oxidase agree with those obtained by JAKOBY AND NIRENBERG inasmuch as they suggest that acetaldehyde is bound to the enzyme as a thiohemiacetal. These observations would appear to resolve the problem as to whether the oxidation of the aldehyde proceeds by dehydrogenation of the aldehyde hydrate, or by oxidation of the thiohemiacetal to an acetyl-S-enzyme with subsequent hydrolysis to acetate and free enzyme (see ref. 12, p. 34). The second thiol group which appears to be situated at the active center has been implicated in the catalytic mechanism of these enzymes to explain the inhibition of activity by excess aldehyde, the combination of the aldehyde with the second sulfhydryl blocking the enzyme 1~. It has already been pointed out, however, that deviations from linearity of the usual double reciprocal plots are inherent in the rate equations for 2-substrate reactions at high concentrations of either substrate. Consequently, it is quite possible that the second sulfhydryl group has no significance in this respect. The absence of inhibition by excess acetaldehyde when DCIP is the electron acceptor presents circumstantial evidence for this view. ACKNOWLEDGEMENTS

The author wishes to express his appreciation to Dr. V. MASSEY for his advice and encouragement and to Dr. K. DALZlEL for many stimulating discussions. A maintenance grant from the Department of Scientific and Industrial Research is gratefully acknowleged. REFERENCES 1 L. MICHAELIS AND M. MENTEN, Biochem. Z., 49 (1913) 338. 2 G. E. BRIGGS AND J. B. S. HALDANE, Biochem. J., 19 (1925) 333. 8 R. A. ALBERTY, J. Am. Chem. Soc., 75 (1953) 1928. 4 K. DALZIEL, Trans. Farad. Soc., 54 (1958) 1247. 5 C. FRIEDEN, Biochim. Biophys. Acta, 24 (1957) 241. V. MASSE'C, Q. H. GIBSON AND C. VEEGER, Biochem. J., 77 (196o) 341. V. MASSEY, G. PALMER AND R. BENNETT, Biochim. Biophys. Acla, 48 (1961) I. 8 K. DALZIEL, D. WHARDALE AND G. PALMER, unpublished experiments. G. PALMER, Biochim. Biophys. Acta, 56 (1962) 444. 10 V. MASSEY, Biochim. Biophys. Acla, 34 (1959) 255. 11 H. R. MAHLER AND D. ELOWE, J. Biol. Chem., 21o (1954) 149. 1~ M. D i x o n AND E. S. WEBB, in Enzymes, Longnlans Green and Co., London, 1958. is H. R. MAHLER, B. MACKLER, D. E. GREEN AND R. BOCK, J. Biol. Chem., 21o (1954) 465 • i, p. D. BOYER, in P. D. BOYER, H. LARDY AND I(. MYRBACK, The Enzymes, Vol. i, 2rid Ed., Academic Press, Inc., New York, 1959, p. 511. 15 A. P. NYGAARD AND H. THEORELL, Acta Chem. Scand., 9 (1955) 13oo. xe M. W. NIRENBERG AND W. B. JAKOBY, Proc. Natl. Acad. Sci. U.S., 46 (196o) 256. 1~ A. O. M, STOPPANI AND C. MILSTEIN, Rev. Soc. Argent. Biol., 33 (1957) 8o. 18 A. O. M. STOPPANI AND C. ~VIILSTEIN, Rev. Soc. Argent. Biol., 33 (1957) loo. 19 A. O. M. STOPPANI AND C. MILSTEIN, Rev. Soc. Argent. Biol., 33 (1957) 149. ~0 A. O. •. STOPPANI AND C. MILSTEIN, Rev. Soc. Argent. Biol., 33 (1957) 289. ~x j. VAN-EYs, A. SAN PIETRO AND N. O. KAPLAN, Science, 127 (1958) 443.

Biochim. Biophys. Acta, 64 (1962) I 3 5 - I 4 8