d-amino acid oxidase iii. effect of ph

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 65164

D-AMINO ACID OXIDASE

I I I . EFFECT OF pH

383

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 October i 3 t h , i964)

SUMMARY

I. The effect of pH 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 pH curves with saturating substrate concentrations (V against pH) may differ considerably from those obtained by the common practice of using fixed substrate concentrations, which are not sufficient to saturate the enzyme at all pH 's (v against pH).

3. The four substrates give markedly different V - p H curves. 4. The effects of pH on Km are very similar for n-alanine and D-methionine,

showing two pK'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 pH on Ki for an aliphatic and an aromatic acid which act as competitive inhibitors show a single pK of 8.5 in the free enzyme and none in the enzyme-inhibitor complex.

INTRODUCTION

The influence of pH 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 pH 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 pH 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 pH on V and on Km

* P r e s e n t address : Norsk H y d r o ' s I n s t i t u t e for Cancer Research , 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. I t 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 pK 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. I t 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 D- methionine is the most rapidly oxidized of all the substrates, apart from o-proline,

Biochim. Biophys. Acta, 96 (1965) 383-389

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D-AMINO ACID OXlDASE :EFFECT OF pH 385

Fig. i. p H curves of D-amino acid oxidase wi th D-alanine. The curve for v was obtained wi th a cons tan t concentrat ion of D-alanine of 5" lO-3 M. Each poin t on the curve for the m a x i m u m velocity V was obtained f rom a separate L ineweaver -Burk plot. Tempera ture 25 °.

Fig. 2. p H curves (max imum velocity) of D-amino acid oxidase wi th D-methionine, glycine and D-norvaline.

(Fig. I a of the preceding paper) and glycine is a very poor substrate in comparison ; yet at pH IO glycine is a bet ter substrate than D-methionine, and is actually oxidized more than half as fast as is D-methionine at pH 8. 5. At lower concentrations, however, glycine is still a poor substrate compared with methionine, even at pH IO, because of its low affinity, I/Km for methionine at this pH being about a thousand times greater than that for glycine. I t 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 pH 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 unex- pected, 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


4

3

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O-met hionine

I I

D-nor'voline I | I I I

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Glycine I I I I

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 fa t ty 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 be- haviour should give further information about the ionization of groups in that site. In some ways the interpretation of pH effects on inhibitors is simpler and more definite than that of pH 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 pH for one acid of each series, with D-alanine as substrate.

DISCUSSION

The theory of pH effects on enzymes has been discussed in some detail by DIXON ~, by ALBERTY 8, and by 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 pK '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 pK's ; upward bends in the -- log Km or -- log Ki curves (appearing also as downward bends in the log V curves) indicate pK ' s of the complexes, and downward bends (not appearing in the log V curves) indicate pK '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


D-AMINO ACID OXIDASE:EFFECT OF pH 387

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Fig. 4. pH-dependence of the affinities of two competitive inhibitors for D-amino acid oxidase.

Fig. 5- pH 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 may 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 pK at about 8.6 for the flee enzyme (since the free inhibitors have no pK 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 fa t ty 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 pH 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 pK ' 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 pK in the free enzyme at about 8.6 as was already shown by the inhibitors. Since no further pK ' s in the free enzyme are revealed by the inhibitor curves, the additional pK is presumably in the free amino acid, and in fact would be ex- pected, since the amino group has a pK in the neighbourhood of 9.7.

The main differences between the curves appear to be in the pK 's for the enzyme-substrate complexes. Those for D-alanine and D-methionine in Fig. 3 indicate two pK'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 pH 5 and I I



the log V curve shows a change in slope of 2 units ( + I to - - I ) , corresponding to the two pK ' 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 pK at about I0, which occurs in the enzyme-amino acid complex but not in the enzyme-fa t ty 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 may throw some light on the mechanism of activation. Specu- lation 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 may be noted that the main difference between the curve for glycine and those for alanine and methionine is the much higher value of this pK in the former case. In any event for curves of this kind, with pK '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 pK '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 pK ' 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. I t fails to account for either the unsym- metrical form of the log V curve for alanine, or for the marked differences between the amino acids. Since the pK'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 mat ter of p K values. The most likely explanation is that the large differences in reaction velocity, due to the specificity of the enzyme, may cause different steps in the reaction sequence to become rate-determining, and that these steps do not depend on pH 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. I t is even possible that different steps might become limiting in different parts of the same pH 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.


D-AMINO ACID OXIDASE :EFFECT OF pH 389

3 V. MASSlSY, G. PALMER AND R. BENNETT, Biochim. Biophys. Acta, 48 (1961) i. 4 M. DxxoN AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 358. 5 M. DIXON AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 357. 6 G. R. BARTLETT, J. Am. Chem. Soc., 7 o (1948) iOlO. 7 M. DixoN, Biochem. J., 55 (1953) 161. 8 R. A. ALBERTY, Enzyme Models and Enzyme Structure, B r o o k h a v e n Nat iona l Labora to ry ,

Up ton , :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.


d-amino acid oxidase iii. effect of ph

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