Malcolm Dixon

d-amino acid oxidase II. Specificity, competitive inhibition and reaction sequence

Abstract 1. 1. A systematic study has been made of the specificity of purified d -amino acid oxidase ( d -amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) of pig kidney, with measurements of V and Km for each amino acid substrate. 2. 2. The enzyme has a high degree of specificity for O2 as hydrogen acceptor; a slight reaction occurs with certain dyes, but none with ferricyanide, cytochrome c, quinones, phenazine methosulphate or NAD. 3. 3. In the activated state the d -amino acids show very large differences in reactivity, quite apart from any differences in affinity for the enzyme. The substrate with by far the highest value of V is d -proline. Among the other substrates the most reactive are d -methionine, d -alanine, d -norleucine, d -isoleucine and d -phenylalanine. Acidic and basic amino acids do not react, with the exception of d -ornithine. The weakly basic d -histidine reacts, as do all ring-containing d -amino acids. The enzyme is completely d specific. 4. 4. The enzyme is competitively inhibited by straight-chain fatty acids. For these the value of 1 K i (the affinity) rises to a maximum with 5 C-atoms, thereafter falling off with increased chain-length. This parallels the effect of chain-length on 1 K m for the corresponding aliphatic amino acid substrates, which suggests that 1 K m gives an indication of substrate affinity. 5. 5. The amines of the amino acids and most of the l -amino acids fail to inhibit; 2-oxo acids and 2-hydroxy acids are stronger inhibitors than fatty acids. It may be inferred that the carboxyl group is essential for combination but the amino group is not, that a suitable aliphatic group or ring makes a considerable contribution to the affinity, and that an amino group in the wrong configuration has a negative affinity which may overcome the positive affinities of the other groups. 6. 6. The effect of varying O2 concentration supports a reaction sequence in which the product dissociates from the enzyme before the reaction with O2 takes place.

Enzyme Fractionation By Salting-Out: A Theoretical Note

Publisher Summary This chapter explores that for many years salting-out by high concentrations of ammonium sulfate has been one of the classical methods of protein separation. The underlying assumption in most cases seems to have been that the different proteins are precipitated at different fixed ammonium sulfate concentrations, provided the pH and temperature are fixed. Unlike fractionation with organic solvents, it does not tend to inactivate enzymes by denaturation; on the contrary, ammonium sulfate frequently has a protective action on enzymes, and indeed enzymes are frequently stored in the form of suspensions of precipitates in concentrated ammonium sulfate. This not only stabilizes the enzyme but prevents the growth of bacteria. Unlike chromatographic and absorption methods, it affords a procedure for concentrating as well as purifying the enzyme. The chapter stresses on the importance of pH and temperature as variables that have a great effect on the positions and separation of the precipitation peaks. They must therefore be carefully controlled. It highlights that a single protein may crystallize in several different forms with different solubilities and the solubility, and hence the composition of the precipitate may change in a complicated manner with time.

D-AMINO ACID OXIDASE. I. DISSOCIATION AND RECOMBINATION OF THE HOLOENZYME.

Abstract 1. 1. The purified d -amino acid oxidase ( d -amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) of pig kidney has been studied by means of the O2 electrode, as a preliminary to a systematic study of its specificity. 2. 2. This technique revealed changes in activity in the early stages after the addition of enzyme to the test solution; these are not observable by the usual manometric method. They are shown to be due to spontaneous dissociation of the holoenzyme into FAD and apoenzyme on dilution. 3. 3. The dissociation and association are not instantaneous, but require several minutes. The rate constant for the dissociation was found to be 0.45 min−1, and that for the combination 1.8·106 M−1 min− at 25° and pH 8.5. The rates are decreased when the enzyme combines with subtrates or competitive inhibitors. 4. 4. The equilibrium constant deduced from these values is 2.5·10−7 M. The value calculated from equilibrium concentrations was 2.8·10−7 M, in good agreement with previous values. 5. 5. The rate of inhibition of the enzyme by p-chloromercuribenzoate is identical with the rate at which FAD dissociates from the enzyme, and is slowed to about the same extent by competitive inhibitors.

The acceptor specificity of flavins and flavoproteins. I. Techniques for anaerobic spectrophotometry.

1. Easily constructed apparatus is described for spectrophotometry under strictly anaerobic conditions without requiring special cuvettes. It permits the addition of several reagents successively without opening the system to the air. 2. The absorption spectrum of dithionite shows a strong peak at 314 nm, the molar absorbance of which has been determined. This gives a convenient method for the titration of acceptors with dithionite. 3. One molecule of dithionite reacts very rapidly with one molecule of O2 in solution. The O2 is reduced quantitatively to H2O2. With excess of dithionite another, much slower, reaction follows, in which a second molecule of dithionite is oxidized by the peroxide. 4. A study has been made of the reduction by dithionite of a variety of acceptors commonly used in the study of flavoproteins. The majority react very rapidly, but a few are reduced relatively slowly or not at all. 5. The majority of acceptors do not react significantly with sulphite, the oxidation product of dithionite. One molecule of dithionite then provides two reduction equivalents. A few acceptors, however, react with the sulphite formed, giving a second reaction involving two more equivalents.

The acceptor specificity of flavins and flavoproteins. III. Flavoproteins

Abstract 1. The specificity of flavoproteins towards acceptors has been rather neglected, but an attempt is here made to construct a comparative table of acceptor specificities of those flavoprotein enzymes for which data exist. 2. The acceptor specificity of reduced flavin groups, when combined with apoenzyme proteins, is quite different from that of the same flavin groups in the free state (see Part II). Free flavins react very rapidly with a wide range of acceptors, but the same groups combined as flavoproteins have a severely restricted range of action. 3. There are remarkable differences between different flavoproteins. Nearly every flavoprotein fails altogether to react with at least one, and often several, of the acceptors, giving a specificity pattern which is different in each case. There seems to be no general acceptor for flavoproteins. 4. The effect of combination of a flavin with a particular apoenzyme is to inhibit specifically the reaction of the flavin with particular acceptors with which it would react very rapidly in the absence of the apoenzyme. 5. Each apoenzyme produces its own distinctive pattern of inhibitions. The degree of inhibition is often very high; the table shows over 50 cases of specific inhibitions that are essentially complete. Some of these are very difficult to explain. 6. There is no obvious parallelism between any acceptor and any other in its pattern of reactivity with a series of different flavoproteins. 7. In a few cases combination with apoenzyme specifically accelerates the reaction of the flavin with particular acceptors, so that the flavoprotein is oxidized faster than the free flavin. 8. Possible correlations are discussed between the effects of apoenzymes on the reactivity of flavins with acceptors and a number of special known features of different apoenzymes, but no adequate explanation of the differences in specificity has emerged. 9. In view of the interesting nature of the effects, a plea is made for a more intensive study of the acceptor side of flavoprotein specificity.

D-aspartate oxidase of kidney.

1. 1.|The d-aspartate oxidase (d-aspartate:oxygen oxidoreductase (deaminating), EC 1.4.3.1.) of rabbit kidney has been purified almost 100-fold. Although it is not yet homogeneous, it is apparently free from other oxidizing enzymes. 2. 2.|The enzyme is a flavoprotein. As prepared, it is in the inactive apo-form. The activity is restored by adding FAD, but not by FMN or riboflavin. The combination and dissociation of FAD are not instantaneous, but require about 15 min at 30° for approximate completion. 3. 3.|The enzyme oxidizes d-aspartate and d-glutamate, but has no action on any of the substrates of d-amino-acid oxidase or on any l-amino acids. d-Aspartate and d-glutamate are oxidized by the same enzyme. At low concentrations, using ferricyanide as acceptor, d-aspartate is oxidized more rapidly than d-glutamate, but at high concentrations the reverse is the case. With oxygen as acceptor, however, d-aspartate is always more rapidly oxidized than d-glutamate. 4. 4.|Unlike d-amino-acid oxidase, d-aspartate oxidase is strongly and competitively inhibited by dicarboxylic but not by monocarboxylic acids. 5. 5.|Only three acceptors have been found to work with the enzyme, namely ferricyanide, oxygen and dichlorophenolindophenol. Ferricyanide, which is inactive with d-amino-acid oxidase, is the best acceptor. With ferricyanide or oxygen, the rate decreases with increase of acceptor concentration; with indophenol it increases. 6. 6.|With ferricyanide as acceptor, the progress curve of the enzyme reaction is autocatalytic in form. The increase of the velocity with time is due to the fall in the ferricyanide concentration as the reaction proceeds, and the consequent reversal of the inhibition by ferricyanide. 7. 7.|The pH optimum is exceptionally sharp; the fall on the alkaline side is due to destruction of the enzyme. The position of the optimum depends on the order of addition of the reactants; if the ferricyanide is added last it is at about pH 9.6, if the substrate is added last it is at 8.7. The effect of pH on the Km for d-aspartate is complex, but the curve is quite different from that of d-amino-acid oxidase.

d-Amino acid oxidase III. Effect of pH

Abstract 1. 1. The effect of pH on V and K m 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 for four substrates. 2. 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 pHs ( v against pH). 3. 3. The four substrates give markedly different V -pH curves. 4. 4. The effects of pH on K m are very similar for d -alanine and d -methionine, showing two p K s of about 7.5 and 10 in the enzyme-substrate complex, one of 8.5 in the free enzyme, and one in the range 9.2–9.8 in the free substrate. The curve for glycine differs somewhat in form, and that for d -norvaline is completely different. 5. 5. The effects of pH on K i 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.

The acceptor specificity of flavins and flavoproteins. II. Free flavins

1. For comparison with flavoprotein oxidases, a study has been made of free flavins in the reduced form with respect to the specificity and stoichiometry of their oxidation by a series of acceptors. 2. Reduced flavins uncombined with proteins show very little acceptor specificity and react very rapidly with nearly all the commonly used acceptors. Their behaviour resembles that of dithionite very closely indeed, and it differs considerably from that of flavoproteins. Like dithionite, free reduced flavins reduce O2 quantitatively to H2O2; this oxidizes a further molecule of flavin. 3. H2O2 and cytochrome c react more slowly than most acceptors with reduced flavins. Nitrate and NDA+ do not act at all and require special activation. 4. Catalase can act as a catalyst for the aerobic oxidation of flavins by converting slowly-reacting H2O2 into rapidly-reacting O2. 5. In the absence of catalytic metals ascorbate reacts with acceptors much more slowly than reduced flavins do.