Affinity labeling of D-amino acid oxidase with an acetylenic substrate.

The acetylenic substrate, D-2-amino-4-pentynoic acid (D-propargylglycine), was oxidatively deaminated by hog kidney D-amino acid oxidase[EC 1.4.3.3], with accompanying inactivation of the enzyme. The flavin which was extracted by hot methanol from the inactivated enzyme was identical with authentic FAD by thin-layer chromatography and circular dichroism. The excitation spectrum of emission at 520 nm of the released flavin was very similar to the absorption spectrum of oxidized FAD. The released flavin was reduced by potassium borohydride. The apoenzyme prepared after propargylglycine treatment did not show restored D-amino acid oxidase activity on adding exogenous FAD. The absorption spectrum of this inactivated apoenzyme showed absorption peaks at 279 and 317 nm, and a shoulder at about 290 nm. These results strongly indicate that the inactivation reaction is a dynamic affinity labeling with D-propargylglycine which produces irreversible inactivation of the enzyme by a covalent modification of an amino acid residue at the active site.     

Three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase. Optimization of the oxidative half-reaction through alignment of the product with reduced flavin

The three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase (DAO) was solved by cryo-X-ray crystallography; the purple intermediate is known to comprise a complex between the dehydrogenated product, an imino acid, and the reduced form of DAO. The crystalline purple intermediate was obtained by anaerobically soaking crystals of oxidized DAO in a buffer containing excess D-proline as the substrate. The dehydrogenated product, delta(1)-pyrrolidine-2-carboxylate (DPC), is found sandwiched between the phenol ring of Tyr 224 and the planar reduced flavin ring. The cationic protonated imino nitrogen is within hydrogen-bonding distance of the backbone carbonyl oxygen of Gly 313. The carboxyl group of DPC is recognized by the Arg 283 guanidino and Tyr 228 hydroxyl groups through ion-pairing and hydrogen-bonding, respectively. The (+)HN=C double bond of DPC overlaps the N(5)-C(4a) bond of reduced flavin. The electrostatic effect of the cationic nitrogen of DPC is suggested to shift the resonance hybridization of anionic reduced flavin toward a canonical form with a negative charge at C(4a), thereby augmenting the electron density at C(4a), from which electrons are transferred to molecular oxygen during reoxidation of reduced flavin. The reactivity of reduced flavin in the purple intermediate, therefore, is enhanced through the alignment of DPC with respect to reduced flavin.     

Exchange of free and bound coenzyme of flavin enzymes studied with [14C]FAD.

The exchange of bound FAD for free FAD was studied with D-amino acid oxidase (D-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) and beta-D-glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4). For a simple measurement of the reaction rate, equimolar amounts of the enzyme and [14C]FAD were mixed. The exchange occurred very rapidly in the holoenzyme of D-amino acid oxidase at 25 degrees C, pH 8.3 (half life of the exchange: 0.8 min), but slowly in the presence of the substrate or a competitive inhibitor, benzoate. It also occurred slowly in the purple complex of D-amino acid oxidase. In the case of beta-D-glucose oxidase, however, the exchange occurred very slowly at 25 degrees C, pH 5.6, regardless of the presence of the substrate or p-chloromercuribenzoate. On the basis of these findings, the turnover of the coenzymes of flavin enzymes in mammals is discussed.     

Analyzing the D-amino acid content in biological samples by engineered enzymes

The aim of our present research is to produce mutant forms of D-amino acid oxidase from Rhodotorula gracilis in order to determine D-amino acid content in different biological samples. During the past few years, our group has produced yeast D-amino acid oxidase variants with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all D-amino acids) both by rational design and directed evolution methods. Now, the kinetic constants for a number of amino acids (even for unnatural ones) of the most relevant D-amino acid oxidase variants have been investigated. This information constitutes the basis for considering potential analytical applications in this important field.     

Vinylglycine and proparglyglycine: complementary suicide substrates for L-amino acid oxidase and D-amino acid oxidase.

Proparglyglycine (2-amino-4-pentynoate) and vinylglycine (2-amino-3-butenoate) have been examined as substrates and possible inactivators of two flavo enzymes, D-amino acid oxidase from pig kidney and L-amino acid oxidase from Crotalus adamanteus venom. Vinylglycine is rapidly oxidized by both enzymes but only L-amino acid oxidase is inactivated under assay conditions. The loss of activity probably involves covalent modification of an active site residue rather than the flavin adenine dinucleotide coenzyme and occurs once every 20000 turnovers. We have confirmed the recent observation (Horiike, K, Hishina, Y., Miyake, Y., and Yamano, T. (1975) J, Biochem. (Tokyo), 78, 57) that D-proparglglycine is oxidized with a time-dependent loss of activity by D-amino acid oxidase and have examined some mechanistic aspects of this inactivation, The extent of residual oxidase activity, insensitive to further inactivation, is about 2%, at which point 1.7 labels/subunit have been introduced with propargly[2-14C]glycine as substrate. L-Proparglyclycine is a substrate but not an inactivator of L-amino acid oxidase and the product ahat accumulats in the nonnucleophilic N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer is acetopyruvate. In the presence of butylamine HCl, a species with lambdaman 317 nm (epsilon = 15 000) accumulates that may be a conjugated eneamine adduct. The same species accumulates from D-amino acid oxidase oxidation of D-propargylglycine prior to inactivation; the inactivated apo D-amino acid oxidase has a new peak at 317 nm that is probably a similar eneamine. A likely inactivating species is 2-keto-3,4-pentadienoate arising from facile rearrangement of the expected initial product 2-keto 4 pentynoate. Vinylglycine and proparglyglycine show inactivation specificity, then, for L-and D-amino acid oxidase, respectively.     

Kinetic mechanisms of glycine oxidase from Bacillus subtilis.

The kinetic properties of glycine oxidase from Bacillus subtilis were investigated using glycine, sarcosine, and d-proline as substrate. The turnover numbers at saturating substrate and oxygen concentrations were 4.0 s(-1), 4.2 s(-1), and 3.5 s(-1), respectively, with glycine, sarcosine, and D-proline as substrate. Glycine oxidase was converted to a two-electron reduced form upon anaerobic reduction with the individual substrates and its reductive half-reaction was demonstrated to be reversible. The rates of flavin reduction extrapolated to saturating substrate concentration, and under anaerobic conditions, were 166 s(-1), 170 s(-1), and 26 s(-1), respectively, with glycine, sarcosine, and D-proline as substrate. The rate of reoxidation of reduced glycine oxidase with oxygen in the absence of product (extrapolated rate approximately 3 x 10(4) M(-1) x s(-1)) was too slow to account for catalysis and thus reoxidation started from the reduced enzyme:imino acid complex. The kinetic data are compatible with a ternary complex sequential mechanism in which the rate of product dissociation from the reoxidized enzyme form represents the rate-limiting step. Although glycine oxidase and D-amino acid oxidase differ in substrate specificity and amino acid sequence, the kinetic mechanism of glycine oxidase is similar to that determined for mammalian D-amino acid oxidase on neutral D-amino acids, further supporting a close similarity between these two amine oxidases.     

D-氨基酸氧化酶研究进展

D-氨基酸氧化酶(D-amino acid oxidase:oxidoreductase, DAAO, EC 1.4.3.3)是一种以黄素腺嘌呤(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸的氨基生成相应的酮酸和氨。在体内D-氨基酸的代谢中起着重要作用。主要介绍了D-氨基酸氧化酶的生理功能和应用、表达条件优化及通过定点突变对酶学性质的研究。     

Temperature-induced changes in the coenzyme environment of D-amino acid oxidase revealed by the multiple decays of FAD fluorescence.

A temperature-dependent change in the microenvironment of the coenzyme, FAD, of D-amino acid oxidase was investigated by means of steady-state and picosecond time-resolved fluorescence spectroscopy. Relative emission quantum yields from FAD bound to D-amino acid oxidase revealed the temperature transition when concentration of the enzyme was lowered. The observed fluorescence decay curves were well described with four-exponential decay functions. The amplitude of the shortest lifetime (tau 0), approximately 25 ps, was always negative, which indicates that the fluorescence of D-amino acid oxidase at approximately 520 nm appears after a metastable state of the excited isoalloxazine decays. The other components with positive amplitudes were assigned to dimer or associated forms of the enzyme, monomer, and free FAD dissociated from the enzyme. Ethalpy and entropy changes of intermediate states in the quenching processes were evaluated according to the absolute rate theory. The temperature transition was much more pronounced in the monomer than in the dimer or associated forms of the enzyme.     

The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis

The role of the active site hydrogen bond of cytochrome P-450cam has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450cam normally catalyzes the regiospecific hydroxylation of the monoterpene camphor. The x-ray crystal structure of this soluble bacterial cytochrome P-450 (Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16128) indicates a specific hydrogen bond between tyrosine 96 and the carbonyl moiety of the camphor substrate. The site-directed mutant in which tyrosine 96 has been changed to a phenylalanine and the substrate analogues thiocamphor and camphane have been used to probe this interaction in several aspects of catalysis. At room temperature, both the mutant enzyme with camphor and the wild type enzyme with thiocamphor bound result in 59 and 65% high-spin ferric enzyme as compared to the 95% high spin population obtained with native enzyme and camphor as substrate. The equilibrium dissociation constant is moderately increased, from 1.6 microM for the wild type protein to 3.0 and 3.3 microM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450cam exhibits a larger decrease in high spin fraction (45%) and a correspondingly larger KD (46 microM), suggesting that the carbonyl moiety of camphor plays an important steric role in addition to its interaction as a hydrogen bond acceptor. The absolute regioselectivity of the mutant enzyme, and of the wild type enzyme with thiocamphor, is lost resulting in production of several hydroxylated products in addition to the 5-exo-hydroxy isomer. Based on rates of NADH oxidation, comparison of the substrate specificity for these systems (kcat/KD) indicates a 5- and 7-fold decrease in specificity for the mutant enzyme and thiocamphor-wild type complex, respectively. The replacement of the cytochrome P-450cam active site tyrosine with phenylalanine does not affect the branching ratio of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J. Am. Chem. Soc. 109, 3754-3760).     

Yeast D-amino acid oxidase: structural basis of its catalytic properties

The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9A) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF(3)-alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2-C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.     

Interaction of steroids with D-amino acid oxidase.

1. Progesterone inhibited D-amino acid oxidase (D-amino acid : O2 oxidoreductase (deaminating), EC 1.4.3.3) in competition with its substrate, D-alanine. Binding of progesterone brought about the increase in both fluorescence intensity and fluorescence polarization of FAD, which indicates that the environment surrounding FAD chromophore is modified due to a conformational change in the apoenzyme. 2. Ethinyl estradiol, testosterone, testosterone propionate, corticosterone and aldosterone also inhibited the enzyme slightly in the same manner. Their binding also produced a slight increase in FAD fluorescence without decreasing the fluorescence polarization. 3. Cholesterol did not inhibit the enzyme, though it increased the fluorescence polarization of FAD. This indicates the binding of cholesterol with the enzyme at a site other than the substrate binding site.     

Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.     

Detection and substrate selectivity of new microbial D-amino acid oxidases

In order to screen for new microbial D-amino acid oxidase activities a selective and sensitive peroxidase/o-dianisidine assay, detecting the formation of hydrogen peroxide was developed. Catalase, which coexists with oxidases in the peroxisomes or the microsomes and, which competes with peroxidase for hydrogen peroxide, was completely inhibited by o-dianisidine up to a catalase activity of 500 nkat ml(-)(1). Thus, using the peroxidase/o-dianisidine assay and employing crude extracts of microorganisms in a microplate reader, a detection sensitivity for oxidase activity of 0.6 nkat ml(-)(1) was obtained.Wild type colonies which were grown on a selective medium containing D-alanine as carbon, energy and nitrogen source were examined for D-amino acid oxidase activity by the peroxidase/o-dianisidine assay. The oxidase positive colonies possessing an apparent oxidase activity > 2 nkat g dry biomass(-)(1) were isolated. Among them three new D-amino acid oxidase-producers were found and identified as Fusarium oxysporum, Verticilium lutealbum and Candida parapsilosis. The best new D-amino oxidase producer was the fungus F. oxysporum with a D-amino acid oxidase activity of about 900 nkat g dry biomass(-)(1) or 21 nkat mg protein(-)(1). With regard to the use as a biocatalytic tool in biotechnology the substrate specificities of the three new D-amino acid oxidases were compared with those of the known D-amino acid oxidases from Trigonopsis variabilis, Rhodotorula gracilis and pig kidney under the same conditions. All six D-amino acid oxidases accepted the D-enantiomers of alanine, valine, leucine, proline, phenylalanine, serine and glutamine as substrates and, except for the D-amino acid oxidase from V. luteoalbum, D-tryptophane, D-tyrosine, D-arginine and D-histidine were accepted as well. The relative highest activities (>95%) were measured versus D-alanine (C. parapsilosis, F. oxysporum, T. variabilis), D-methionine (V. luteoalbum, R. gracilis), D-valine (T. variabilis, R. gracilis) and D-proline (pig kidney). The D-amino oxidases from F. oxysporum and V. luteoalbum were able to react with the industrially important substrate cephalosporin C although the D-amino acid oxidase from T. variabilis was at least about 20-fold more active with this substrate.As the results of our studies, a reliable oxidase assay was developed, allowing high throughput screening in a microplate reader. Furthermore, three new microbial D-amino acid oxidase-producers with interesting broad substrate specificities were introduced in the field of biotechnology.     

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

Summary The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.     

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.     

pH and kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase.

Primary deuterium kinetic isotope and pH effects on the reduction of D-amino acid oxidase by amino acid substrates were determined using steady-state and rapid reaction methods. With D-serine as substrate, reduction of the enzyme-bound FAD requires that a group with a pKa value of 8.7 be unprotonated and that a group with a pKa value of 10.7 be protonated. The DV/Kser value of 4.5 is pH-independent, establishing that these pKa values are intrinsic. The limiting rate of reduction of the enzyme shows a kinetic isotope effect of 4.75, consistent with this as the intrinsic value. At high enzyme concentration (approximately 15 microM) at pH 9,D-serine is slightly sticky (k3/k2 = 0.8), consistent with a decrease in the rate of substrate dissociation. With D-alanine as substrate, the pKa values are perturbed to 8.1 and 11.5. The DV/Kala value increases from 1.3 at pH 9.5 to 5.1 at pH 4, establishing that D-alanine is sticky with a forward commitment of approximately 10. The effect of pH on the DV/Kala value is consistent with a model in which exchange with solvent of the proton from the group with pKa 8.7 is hindered and is catalyzed by H2O and OH- above pH 7 and by H3O+ and H2O below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The DV/Kgly value increases from 1.26 at pH 6.5 to 3.1 at pH 10.7, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At pH 10.5, the kinetic isotope effect on the limiting rate of reduction is 3.4.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

Thiazolidine-2-carboxylic acid, an adduct of cysteamine and glyoxylate, as a substrate for D-amino acid oxidase

A mixture of cysteamine and glyoxylate, proposed by Hamilton et al. to form the physiological substrate of hog kidney D-amino acid oxidase (Hamilton, G. A., Buckthal, D. J., Mortensen, R. M., and Zerby, K. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2625-2629), was confirmed to act as a good substrate for the pure enzyme. As proposed by those workers, it was shown that the actual substrate is thiazolidine-2-carboxylic acid, formed from cysteamine and glyoxylate with a second order rate constant of 84 min-1 M-1 at 37 degrees C, pH 7.5. Steady state kinetic analyses reveal that thiazolidine-2-carboxylic acid is a better substrate at pH 8.5 than at pH 7.5. At both pH values, the catalytic turnover number is similar to that obtained with D-proline. D-Amino acid oxidase is rapidly reduced by thiazolidine-2-carboxylic acid to form a reduced enzyme-imino acid complex, as is typical with D-amino acid oxidase substrates. The product of oxidation was shown by NMR to be delta 2-thiazoline-2-carboxylic acid. Racemic thiazolidine-2-carboxylic acid is completely oxidized by the enzyme. The directly measured rate of isomerization of L-thiazolidine-2-carboxylic acid to the D-isomer was compared to the rate of oxidation of the L-isomer by D-amino acid oxidase. Their identity over the range of temperature from 2-30 degrees C established that the apparent activity with the L-amino acid can be explained quantitatively by the rapid, prior isomerization to D-thiazolidine-2-carboxylic acid.     

Effect of hydrophobic probes on the higher structure of D-amino acid oxidase.

1. The holoenzyme of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] was found to combine with 1-anilinonaphthalene-8-sulfonate without liberation of its coenzyme, FAD. No energy transfer interaction was found to occur between the bound dye and FAD of the holoenzyme. On the other hand, when the apoenzyme was bound to the dye and then to FAD, energy transfer interaction between the bound dye and bound FAD was observed. In both cases, the dye competes with the substrate, D-alanine. It is concluded that the dye bound to the holoenzyme is oriented in such a special manner that the mutual orientation factor between the dye and FAD becomes very small in magnitude. 2. When the apoenzyme combined with the dye, the monomer-dimer equilibrium of the apoenzyme shifted towards the dimer. On the other hand, 4-monobenzoylamido-4'-aminostilbene-2,2'-disulfonate combined with the apoenzyme to induce monomerization.     

Lifetime of the purple intermediate of D-amino acid oxidase under anaerobic conditions.

The lifetime of the purple intermediate formed from D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] and a neutral D-amino acid under anaerobic conditions was measured with a series of neutral D-amino acids. The lifetime increases with increase in the number of carbons in the side chain of the amino acid up to 4-5 atoms and then decreases with further increase in the number of carbons. This suggests that the hydrophobicity of the alkyl group of the neutral D-amino acid determines the lifetime of the purple intermediate unless a steric effect occurs.