- Email: [email protected]
d -Amino acid oxidase III. Effect of pH
BIOCHIMICA ET BIOPHYSICA ACTA
383
BBA 65164 D-AMINO ACID O X I D A S E I I I . E F F E C T OF p H
M A L C O L M D I X O N AND K J E L L K L E P P E *
Department of Biochemistry, University of Cambridge, Cambridge (Great Britain) (Received O c t o b e r i 3 t h , i964)
SUMMARY
I. The effect of p H on V and Km for the action of purified D-amino acid oxidase (D-amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3) of pig kidney has been studied fo:r four substrates. 2. The p H curves with saturating substrate concentrations (V against pH) m a y differ considerably from those obtained b y the common practice of using fixed substrate concentrations, which are not sufficient to saturate the enzyme at all p H ' s (v against pH). 3. The four substrates give markedly different V - p H curves. 4. The effects of p H on Km are very similar for n-alanine and D-methionine, showing two p K ' s of about 7.5 and IO in the enzyme-substrate complex, one of 8.5 in the flee: enzyme, and one in the range 9.2<).8 in the free substrate. The curve for glycine differs somewhat in form, and that for D-norvaline is completely different. 5. The effects of p H on Ki for an aliphatic and an aromatic acid which act as competitive inhibitors show a single p K of 8.5 in the free enzyme and none in the enzyme-inhibitor complex.
INTRODUCTION
The influence of p H on conjugated enzymes is a somewhat complex matter, but usually it can be discussed in terms of dissociations of the enzyme itself and effects on the kinetic constants V and Km of particular substrates. Among flavoproteins, information on the effects of p H on the latter constants is meagre, because the rate has been determined hitherto at an arbitrary substrate concentration; thus it is not possible to say whether any given effect of p H is on the reactivity or on the affinity of the substrate in question. D-Amino acid oxidase (D-amino acid :O~ oxidoreductase (deaminating), EC 1.4.3.3) is especially suitable for investigating the effect of p H on V and on Km * P r e s e n t a d d r e s s : N o r s k H y d r o ' s I n s t i t u t e for Cancer R e s e a r c h , Oslo (Norway).
Biochim. Biophys. Acta, 96 (I965) 383-389
384
M. DIXON, K. KLEPPE
because it has a wide specificity for D-amino acids, and thus one can compare the effects with a number of different substrates. Earlier studies on the effect of pH on this enzyme 1,2 have dealt with its influence on the thermostability of the enzyme and on the velocity measured at one arbitrary substrate concentration. Since it is now possible to obtain the holoenzyme in a pure form 3, and since V and Km have been measured separately at pH 8.5 for many substrates, as described in the preceding paper 4, it was decided to investigate the effect o f p H on v, V, Km and Ki for a number of substrates and competitive inhibitors. It has been found that the different substrates have somewhat different pH optima for V. The effects of pH on V, Km and Kl lead to the conclusion that the enzyme-substrate complex usually has pK's in the region of pH 7.5 and IO, not seen in the free enzyme or substrate, and that the free enzyme has a group in or near the active centre with a p K of about 8. 5. MATERIALS AND METHODS
The materials were as described in the preceding paper' and the enzyme was prepared by the method of MASSEY et al. 3 as described in the first paper of this series 5. The buffers used were as follows: for pH 5.0-8.0, 0.05 M phosphate-citrate; for pH 8.O-lO.O, 0.05 M pyrophosphate; for pH IO.O-ii.o, 0.05 M trimethylamine. The 02 electrode equipment described earlier 5 was used with the same experimental conditions. The total volume of the reaction mixture was 4 ml, which included o.I ml of 5.7" IO-4 M FAD in each case. Buffers saturated with air at 25 ° were used throughout. The pH was read directly in the reaction vessel at the end of the reaction period, using a Radiometer pH meter. Occasionally o.I ml of o . I % catalase (EC 1.11.1.6) was added at the end in order to check that none of the H202 produced had decomposed to 02 and water during the reaction. Km and V were determined from Lineweaver-Burk plots, varying the substrate concentration but keeping the initial 02 concentration constant. RESULTS
Effect of p H on v and V The results of these experiments are shown in Figs. I and 2. V'our amino acids were studied, namely D-alanine, D-methionine, D-norvaline and glycine. Fig. I shows for D-alanine the effect of pH on both the maximum velocity V and on v, the rate at a constant non-saturating substrate concentration. It will be seen that the pH optima for v and V are at different values. These curves illustrate the fact that pH curves obtained (as are most of those to be found in the literature of enzymes) with substrate concentrations which are insufficient to saturate the enzyme at all pH's do not give the true effect of pH on the reactivity of the activated substrate, since they are affected by variations of affinity with pH. Comparison of the V curves for the different amino acids in Fig. 2 shows what large differences may occur with one and the same enzyme and suggests that each amino acid possesses its own characteristic pH optimum. Moreover it is evident that if the specificity study reported in the preceding paper were done at a higher pH value the relative rates would be appreciably different. For example, at pH 8.5 Dmethionine is the most rapidly oxidized of all the substrates, apart from o-proline, Biochim. Biophys. Acta, 96 (1965) 383-389
D-AMINO ACID OXlDASE :EFFECT OF
pH
385 o O-meghi~nine A D-13OPVO r ~ e
x GlycinP
0-aloninc
16 A
T E 12 IE:
E:
~8 E
:a.
/
4
.%
6
7
pH
8
O
10
11
/
pH
Fig. i. p H curves of D-amino acid oxidase w i t h D-alanine. The curve for v was obtained w i t h a c o n s t a n t concentration of D-alanine of 5" lO-3 M. E a c h p o i n t on the curve for the m a x i m u m velocity V was obtained f r o m a separate L i n e w e a v e r - B u r k plot. T e m p e r a t u r e 25 °. Fig. 2. p H curves ( m a x i m u m velocity) of D-amino acid oxidase w i t h D-methionine, glycine and D-norvaline.
(Fig. I a of the preceding paper) and glycine is a very poor substrate in comparison ; yet at p H IO glycine is a better substrate than D-methionine, and is actually oxidized more than half as fast as is D-methionine at p H 8. 5. At lower concentrations, however, glycine is still a poor substrate compared with methionine, even at p H IO, because of its low affinity, I/Km for methionine at this p H being about a thousand times greater than that for glycine. It should be emphasized that all the V curves given here represent actual reaction rates of the enzyme-substrate complexes, with all effects of affinity changes eliminated; thus they show changes in rate constants other than k+l and k_ v
Effect of pH on Km Fig. 3 shows the influence of p H on pKm for the four amino acids. The curves for the two most active substrates, D-methionine and D-alanine are very similar in form, though not identical; that for D-norvaline is very different, while that for glycine is similar in general form to the methionine and alanine curves, but shows a difference in the left hand slope. The interpretation of these results, which is not easy, is discussed later. The difference between D-alanine and D-norvaline is especially remarkable and unexpected, in view of the fact that they have almost identical ionization constants and their structures are so similar that one would expect them to have the same reaction mechanism.
Biochim. Biophys. Acta, 96 (I965) 383-389
386 4
M. DIXON, K. KLEPPE O-met hionine
3 I
I
I
|
I
I
I
I
I
I
I
I
I
I
I
I
D-nor'voline 4 3
I
I
3
I
o-ak3rine
2 I
I
Glycine
pH
Fig. 3. pH-dependence of --log Kna of D-amino acid oxidase with four amino acid substrates. Each point was obtained from a separate Lineweaver-Burk plot. Effect of p H on Ki Both aromatic 6 and f a t t y acids 4 are good competitive inhibitors of D-amino acid oxidase. Since they compete with the amino acid substrate, they must be regarded as combining with the substrate-binding site in the enzyme, and their behaviour should give further information about the ionization of groups in that site. In some ways the interpretation of p H effects on inhibitors is simpler and more definite than that of p H effects on substrates, for I / K i gives the true affinity of the inhibitor for the enzyme. In Fig. 4 the logarithm of the affinity (i.e. - - log Ki) has been plotted against p H for one acid of each series, with D-alanine as substrate.
DISCUSSION The theory of p H effects on enzymes has been discussed in some detail b y DIXON ~, b y ALBERTY8, and b y DIXON AND WEBB ~. The effects are due to changes in the state of ionization of the components of the system, and should be interpretable in terms of the p K ' s of the free enzyme, the free reactants, and the complexes of the enzyme with the reactants. According to theory, the bends in the logarithmic plots take place at the various p K ' s ; upward bends in the --log Km or --log Ki curves (appearing also as downward bends in the log V curves) indicate p K ' s of the complexes, and downward bends (not appearing in the log V curves) indicate p K ' s of free components (enzyme, substrate or inhibitor). Each p K produces a change of I unit in the slope of the curves, but where the p K of a group is the same in the complex as Biochim. Biophys. Acta, 96 (1965) 383-389
D-AMINO ACID OXIDASE:EFFECT OF p H
387
o so¢liurn b e n z o a t e
sodium valerate
D-alanine
E
~3
i r
J
n" 2
~n .Q I
i i
i f] ...... >
1
/
i
i
~o 7
8
9 pH
110 11
5
6
7
pN
8
9
10
11
Fig. 4. pH-dependence of the affinities of two competitive inhibitors for D-amino acid oxidase. Fig. 5- p H curves for --log Km and log V with D-alanine compared. The broken lines represent one possible interpretation, with two ionizations in the enzyme-substrate complex affecting both curves, and two in the free enzyme affecting Km only.
in the free component it will not appear in the Km or K1 curves, for it m a y be regarded as producing upward and downward bends which cancel out. Considering first the curves for the inhibitors (Fig. 4), we note that they show a single p K at about 8.6 for the flee enzyme (since the free inhibitors have no p K in this region), and that this ionization is no longer present in the enzyme-inhibitor complex. This basic group is therefore evidently the one in the enzyme with which the inhibitor combines through its own carboxyl group, the p K of which is well outside the range of the observations. We have already seen that the carboxyl groups of the amino acid substrates are essential for their binding, and no doubt they combine with the same basic group in the enzyme as do the f a t t y acid inhibitors. Indications of a p K in the free enzyme at about the same p H are in fact seen in the curves of Fig. 3. The curves for the inhibitors show a change of slope of - - I unit over the p H range 8-1o, indicating a single pK. The first three curves for the amino acids, on the other hand, are probably to be taken as indicating two p K ' s in the free enzyme or substrate in this range (a change of slope from o to --2 in the case of norvaline, and from a + slope through zero to a -- slope in the other two cases). One of these is no doubt the same p K in the free enzyme at about 8.6 as was already shown b y the inhibitors. Since no further p K ' s in the free enzyme are revealed b y the inhibitor curves, the additional p K is presumably in the free amino acid, and in fact would be expected, since the amino group has a p K in the neighbourhood of 9.7. The main differences between the curves appear to be in the p K ' s for the enzyme-substrate complexes. Those for D-alanine and D-methionine in Fig. 3 indicate two p K ' s for the complex (upward bends). According to theory these should also appear in the log V curves, but those for the free components should not. In Fig. 4 the --log Km and V curves have been plotted on the same scale, and the broken lines suggest an interpretation which fulfils the expectations. Between p H 5 and I I Biochim. Biophys. Acta, 96 (1965) 383-389
388
M. DIXON, K. KLEPPE
the log V curve shows a change in slope of 2 units ( + I to - - I ) , corresponding to the two p K ' s shown for the complex. The same is true for methionine, which gives a rather more symmetrical log V curve corresponding more closely with the broken line. The p K at about I0, which occurs in the enzyme-amino acid complex but not in the e n z y m e - f a t t y acid complex, is possibly that of the amino group, raised in value by about half a unit when the amino acid is activated by combination with the enzyme. I f so, it m a y throw some light on the mechanism of activation. Speculation as to the chemical nature of the group giving the other p K of the complex would not be profitable in the absence of further evidence, but it m a y be noted that the main difference between the curve for glycine and those for alanine and methionine is the much higher value of this p K in the former case. In any event for curves of this kind, with p K ' s fairly close together, the experimental errors allow too much latitude for precision in interpretation to be attainable. The most surprising feature is the large difference shown by norvaline, where both p K ' s of the complex seem to be missing. This is hard to explainl but in view of its chemical similarity to alanine it is unlikely that the two ionizations are completely suppressed by combination with the enzyme, and it is more probable that the p K ' s have been shifted outside the range of the observations. The V curve for this amino acid in Fig. 2 also shows marked differences from the others. Present theory, which was worked out for simpler enzyme systems, is only partially successful in the present case. It fails to account for either the unsymmetrical form of the log V curve for alanine, or for the marked differences between the amino acids. Since the p K ' s of the four amino acids differ only slightly, and the same enzyme is involved in each case, one would expect curves very similar in form (though not necessarily in vertical positioning) for all, and the differences can hardly be solely a matter of p K values. The most likely explanation is that the large differences in reaction velocity, due to the specificity of the enzyme, m a y cause different steps in the reaction sequence to become rate-determining, and that these steps do not depend on p H in the same way. For example, with one substrate the dissociation of product from the enzyme might be the limiting step, while with another it might be the combination of substrate with the enzyme. It is even possible that different steps might become limiting in different parts of the same p H curve as the velocity changes. A fuller interpretation must await the measurement of the velocity constants of the separate steps of this enzyme react'ion.
ACKNOWLEDGEMENTS
We are grateful to the Royal Society for a grant to M. D. for the purchase of the O~ electrode outfit. One of us (K. K.) is indebted to the Norwegian Council for Scientific and Industrial Research for the award of a Research Fellowship. We wish to thank Mr P. KENWORTHY for valuable technical assistance. REFERENCES I H. A. KREBS, Biochem. J., 29 (1935) 162o. 2 E. WALAAS AND O. WALAAS, Acta Chem. Stand., IO (1956) 122.
Biochim. Biophys. Acta, 96 (1965) 383-389
D-AMINO ACID OXIDASE :EFFECT OF
pH
389
V. MASSlSY, G. PALMER AND R. BENNETT, Biochim. Biophys. Acta, 48 (1961) i. M. DxxoN AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 358. M. DIXON AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 357. G. R. BARTLETT, J. Am. Chem. Soc., 7 o (1948) iOlO. M. DixoN, Biochem. J., 55 (1953) 161. R. A. ALBERTY, Enzyme Models and Enzyme Structure, B r o o k h a v e n N a t i o n a l L a b o r a t o r y , U p t o n , :N.Y., 1962, p. 18. 9 M. D i x o n AND E. C. WEBB, Enzymes, L o n g m a n s , I.ondon, 2nd Ed., I964, p. 116.
3 4 5 6 7 8
Biochim. Biophys. Acta, 96 (1965) 383-389
383
BBA 65164 D-AMINO ACID O X I D A S E I I I . E F F E C T OF p H
M A L C O L M D I X O N AND K J E L L K L E P P E *
Department of Biochemistry, University of Cambridge, Cambridge (Great Britain) (Received O c t o b e r i 3 t h , i964)
SUMMARY
I. The effect of p H on V and Km for the action of purified D-amino acid oxidase (D-amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3) of pig kidney has been studied fo:r four substrates. 2. The p H curves with saturating substrate concentrations (V against pH) m a y differ considerably from those obtained b y the common practice of using fixed substrate concentrations, which are not sufficient to saturate the enzyme at all p H ' s (v against pH). 3. The four substrates give markedly different V - p H curves. 4. The effects of p H on Km are very similar for n-alanine and D-methionine, showing two p K ' s of about 7.5 and IO in the enzyme-substrate complex, one of 8.5 in the flee: enzyme, and one in the range 9.2<).8 in the free substrate. The curve for glycine differs somewhat in form, and that for D-norvaline is completely different. 5. The effects of p H on Ki for an aliphatic and an aromatic acid which act as competitive inhibitors show a single p K of 8.5 in the free enzyme and none in the enzyme-inhibitor complex.
INTRODUCTION
The influence of p H on conjugated enzymes is a somewhat complex matter, but usually it can be discussed in terms of dissociations of the enzyme itself and effects on the kinetic constants V and Km of particular substrates. Among flavoproteins, information on the effects of p H on the latter constants is meagre, because the rate has been determined hitherto at an arbitrary substrate concentration; thus it is not possible to say whether any given effect of p H is on the reactivity or on the affinity of the substrate in question. D-Amino acid oxidase (D-amino acid :O~ oxidoreductase (deaminating), EC 1.4.3.3) is especially suitable for investigating the effect of p H on V and on Km * P r e s e n t a d d r e s s : N o r s k H y d r o ' s I n s t i t u t e for Cancer R e s e a r c h , Oslo (Norway).
Biochim. Biophys. Acta, 96 (I965) 383-389
384
M. DIXON, K. KLEPPE
because it has a wide specificity for D-amino acids, and thus one can compare the effects with a number of different substrates. Earlier studies on the effect of pH on this enzyme 1,2 have dealt with its influence on the thermostability of the enzyme and on the velocity measured at one arbitrary substrate concentration. Since it is now possible to obtain the holoenzyme in a pure form 3, and since V and Km have been measured separately at pH 8.5 for many substrates, as described in the preceding paper 4, it was decided to investigate the effect o f p H on v, V, Km and Ki for a number of substrates and competitive inhibitors. It has been found that the different substrates have somewhat different pH optima for V. The effects of pH on V, Km and Kl lead to the conclusion that the enzyme-substrate complex usually has pK's in the region of pH 7.5 and IO, not seen in the free enzyme or substrate, and that the free enzyme has a group in or near the active centre with a p K of about 8. 5. MATERIALS AND METHODS
The materials were as described in the preceding paper' and the enzyme was prepared by the method of MASSEY et al. 3 as described in the first paper of this series 5. The buffers used were as follows: for pH 5.0-8.0, 0.05 M phosphate-citrate; for pH 8.O-lO.O, 0.05 M pyrophosphate; for pH IO.O-ii.o, 0.05 M trimethylamine. The 02 electrode equipment described earlier 5 was used with the same experimental conditions. The total volume of the reaction mixture was 4 ml, which included o.I ml of 5.7" IO-4 M FAD in each case. Buffers saturated with air at 25 ° were used throughout. The pH was read directly in the reaction vessel at the end of the reaction period, using a Radiometer pH meter. Occasionally o.I ml of o . I % catalase (EC 1.11.1.6) was added at the end in order to check that none of the H202 produced had decomposed to 02 and water during the reaction. Km and V were determined from Lineweaver-Burk plots, varying the substrate concentration but keeping the initial 02 concentration constant. RESULTS
Effect of p H on v and V The results of these experiments are shown in Figs. I and 2. V'our amino acids were studied, namely D-alanine, D-methionine, D-norvaline and glycine. Fig. I shows for D-alanine the effect of pH on both the maximum velocity V and on v, the rate at a constant non-saturating substrate concentration. It will be seen that the pH optima for v and V are at different values. These curves illustrate the fact that pH curves obtained (as are most of those to be found in the literature of enzymes) with substrate concentrations which are insufficient to saturate the enzyme at all pH's do not give the true effect of pH on the reactivity of the activated substrate, since they are affected by variations of affinity with pH. Comparison of the V curves for the different amino acids in Fig. 2 shows what large differences may occur with one and the same enzyme and suggests that each amino acid possesses its own characteristic pH optimum. Moreover it is evident that if the specificity study reported in the preceding paper were done at a higher pH value the relative rates would be appreciably different. For example, at pH 8.5 Dmethionine is the most rapidly oxidized of all the substrates, apart from o-proline, Biochim. Biophys. Acta, 96 (1965) 383-389
D-AMINO ACID OXlDASE :EFFECT OF
pH
385 o O-meghi~nine A D-13OPVO r ~ e
x GlycinP
0-aloninc
16 A
T E 12 IE:
E:
~8 E
:a.
/
4
.%
6
7
pH
8
O
10
11
/
pH
Fig. i. p H curves of D-amino acid oxidase w i t h D-alanine. The curve for v was obtained w i t h a c o n s t a n t concentration of D-alanine of 5" lO-3 M. E a c h p o i n t on the curve for the m a x i m u m velocity V was obtained f r o m a separate L i n e w e a v e r - B u r k plot. T e m p e r a t u r e 25 °. Fig. 2. p H curves ( m a x i m u m velocity) of D-amino acid oxidase w i t h D-methionine, glycine and D-norvaline.
(Fig. I a of the preceding paper) and glycine is a very poor substrate in comparison ; yet at p H IO glycine is a better substrate than D-methionine, and is actually oxidized more than half as fast as is D-methionine at p H 8. 5. At lower concentrations, however, glycine is still a poor substrate compared with methionine, even at p H IO, because of its low affinity, I/Km for methionine at this p H being about a thousand times greater than that for glycine. It should be emphasized that all the V curves given here represent actual reaction rates of the enzyme-substrate complexes, with all effects of affinity changes eliminated; thus they show changes in rate constants other than k+l and k_ v
Effect of pH on Km Fig. 3 shows the influence of p H on pKm for the four amino acids. The curves for the two most active substrates, D-methionine and D-alanine are very similar in form, though not identical; that for D-norvaline is very different, while that for glycine is similar in general form to the methionine and alanine curves, but shows a difference in the left hand slope. The interpretation of these results, which is not easy, is discussed later. The difference between D-alanine and D-norvaline is especially remarkable and unexpected, in view of the fact that they have almost identical ionization constants and their structures are so similar that one would expect them to have the same reaction mechanism.
Biochim. Biophys. Acta, 96 (I965) 383-389
386 4
M. DIXON, K. KLEPPE O-met hionine
3 I
I
I
|
I
I
I
I
I
I
I
I
I
I
I
I
D-nor'voline 4 3
I
I
3
I
o-ak3rine
2 I
I
Glycine
pH
Fig. 3. pH-dependence of --log Kna of D-amino acid oxidase with four amino acid substrates. Each point was obtained from a separate Lineweaver-Burk plot. Effect of p H on Ki Both aromatic 6 and f a t t y acids 4 are good competitive inhibitors of D-amino acid oxidase. Since they compete with the amino acid substrate, they must be regarded as combining with the substrate-binding site in the enzyme, and their behaviour should give further information about the ionization of groups in that site. In some ways the interpretation of p H effects on inhibitors is simpler and more definite than that of p H effects on substrates, for I / K i gives the true affinity of the inhibitor for the enzyme. In Fig. 4 the logarithm of the affinity (i.e. - - log Ki) has been plotted against p H for one acid of each series, with D-alanine as substrate.
DISCUSSION The theory of p H effects on enzymes has been discussed in some detail b y DIXON ~, b y ALBERTY8, and b y DIXON AND WEBB ~. The effects are due to changes in the state of ionization of the components of the system, and should be interpretable in terms of the p K ' s of the free enzyme, the free reactants, and the complexes of the enzyme with the reactants. According to theory, the bends in the logarithmic plots take place at the various p K ' s ; upward bends in the --log Km or --log Ki curves (appearing also as downward bends in the log V curves) indicate p K ' s of the complexes, and downward bends (not appearing in the log V curves) indicate p K ' s of free components (enzyme, substrate or inhibitor). Each p K produces a change of I unit in the slope of the curves, but where the p K of a group is the same in the complex as Biochim. Biophys. Acta, 96 (1965) 383-389
D-AMINO ACID OXIDASE:EFFECT OF p H
387
o so¢liurn b e n z o a t e
sodium valerate
D-alanine
E
~3
i r
J
n" 2
~n .Q I
i i
i f] ...... >
1
/
i
i
~o 7
8
9 pH
110 11
5
6
7
pN
8
9
10
11
Fig. 4. pH-dependence of the affinities of two competitive inhibitors for D-amino acid oxidase. Fig. 5- p H curves for --log Km and log V with D-alanine compared. The broken lines represent one possible interpretation, with two ionizations in the enzyme-substrate complex affecting both curves, and two in the free enzyme affecting Km only.
in the free component it will not appear in the Km or K1 curves, for it m a y be regarded as producing upward and downward bends which cancel out. Considering first the curves for the inhibitors (Fig. 4), we note that they show a single p K at about 8.6 for the flee enzyme (since the free inhibitors have no p K in this region), and that this ionization is no longer present in the enzyme-inhibitor complex. This basic group is therefore evidently the one in the enzyme with which the inhibitor combines through its own carboxyl group, the p K of which is well outside the range of the observations. We have already seen that the carboxyl groups of the amino acid substrates are essential for their binding, and no doubt they combine with the same basic group in the enzyme as do the f a t t y acid inhibitors. Indications of a p K in the free enzyme at about the same p H are in fact seen in the curves of Fig. 3. The curves for the inhibitors show a change of slope of - - I unit over the p H range 8-1o, indicating a single pK. The first three curves for the amino acids, on the other hand, are probably to be taken as indicating two p K ' s in the free enzyme or substrate in this range (a change of slope from o to --2 in the case of norvaline, and from a + slope through zero to a -- slope in the other two cases). One of these is no doubt the same p K in the free enzyme at about 8.6 as was already shown b y the inhibitors. Since no further p K ' s in the free enzyme are revealed b y the inhibitor curves, the additional p K is presumably in the free amino acid, and in fact would be expected, since the amino group has a p K in the neighbourhood of 9.7. The main differences between the curves appear to be in the p K ' s for the enzyme-substrate complexes. Those for D-alanine and D-methionine in Fig. 3 indicate two p K ' s for the complex (upward bends). According to theory these should also appear in the log V curves, but those for the free components should not. In Fig. 4 the --log Km and V curves have been plotted on the same scale, and the broken lines suggest an interpretation which fulfils the expectations. Between p H 5 and I I Biochim. Biophys. Acta, 96 (1965) 383-389
388
M. DIXON, K. KLEPPE
the log V curve shows a change in slope of 2 units ( + I to - - I ) , corresponding to the two p K ' s shown for the complex. The same is true for methionine, which gives a rather more symmetrical log V curve corresponding more closely with the broken line. The p K at about I0, which occurs in the enzyme-amino acid complex but not in the e n z y m e - f a t t y acid complex, is possibly that of the amino group, raised in value by about half a unit when the amino acid is activated by combination with the enzyme. I f so, it m a y throw some light on the mechanism of activation. Speculation as to the chemical nature of the group giving the other p K of the complex would not be profitable in the absence of further evidence, but it m a y be noted that the main difference between the curve for glycine and those for alanine and methionine is the much higher value of this p K in the former case. In any event for curves of this kind, with p K ' s fairly close together, the experimental errors allow too much latitude for precision in interpretation to be attainable. The most surprising feature is the large difference shown by norvaline, where both p K ' s of the complex seem to be missing. This is hard to explainl but in view of its chemical similarity to alanine it is unlikely that the two ionizations are completely suppressed by combination with the enzyme, and it is more probable that the p K ' s have been shifted outside the range of the observations. The V curve for this amino acid in Fig. 2 also shows marked differences from the others. Present theory, which was worked out for simpler enzyme systems, is only partially successful in the present case. It fails to account for either the unsymmetrical form of the log V curve for alanine, or for the marked differences between the amino acids. Since the p K ' s of the four amino acids differ only slightly, and the same enzyme is involved in each case, one would expect curves very similar in form (though not necessarily in vertical positioning) for all, and the differences can hardly be solely a matter of p K values. The most likely explanation is that the large differences in reaction velocity, due to the specificity of the enzyme, m a y cause different steps in the reaction sequence to become rate-determining, and that these steps do not depend on p H in the same way. For example, with one substrate the dissociation of product from the enzyme might be the limiting step, while with another it might be the combination of substrate with the enzyme. It is even possible that different steps might become limiting in different parts of the same p H curve as the velocity changes. A fuller interpretation must await the measurement of the velocity constants of the separate steps of this enzyme react'ion.
ACKNOWLEDGEMENTS
We are grateful to the Royal Society for a grant to M. D. for the purchase of the O~ electrode outfit. One of us (K. K.) is indebted to the Norwegian Council for Scientific and Industrial Research for the award of a Research Fellowship. We wish to thank Mr P. KENWORTHY for valuable technical assistance. REFERENCES I H. A. KREBS, Biochem. J., 29 (1935) 162o. 2 E. WALAAS AND O. WALAAS, Acta Chem. Stand., IO (1956) 122.
Biochim. Biophys. Acta, 96 (1965) 383-389
D-AMINO ACID OXIDASE :EFFECT OF
pH
389
V. MASSlSY, G. PALMER AND R. BENNETT, Biochim. Biophys. Acta, 48 (1961) i. M. DxxoN AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 358. M. DIXON AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 357. G. R. BARTLETT, J. Am. Chem. Soc., 7 o (1948) iOlO. M. DixoN, Biochem. J., 55 (1953) 161. R. A. ALBERTY, Enzyme Models and Enzyme Structure, B r o o k h a v e n N a t i o n a l L a b o r a t o r y , U p t o n , :N.Y., 1962, p. 18. 9 M. D i x o n AND E. C. WEBB, Enzymes, L o n g m a n s , I.ondon, 2nd Ed., I964, p. 116.
3 4 5 6 7 8
Biochim. Biophys. Acta, 96 (1965) 383-389