X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: insight into the role of calcium and surrounding amino acids in active site geometry and catalysis

Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thuringiensis to 2.1 A resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca(2+) ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.     

An anion binding site in the active centre of phospholipase C from Bacillus cereus

The bi-Zn2+-enzyme phospholipase C (Bacillus cereus) is readilly inhibited by univalent anions. N.m.r. studies on the 113Cd-substituted enzyme showed the presence of an inert and a perturbable metal, neither of which seemed affected by I-. X-ray crystallographic analysis showed the binding of one I- to the enzyme 4.8 A from the nearest metal (too far for a metal-halide bond). Phospholipase C contains an arginine residue apparently necessary for substrate binding and I- partially protected against inactivation by an arginine reagent. Thus an arginine residue may represent the binding site for univalent anions in the enzyme active centre.     

Metal ion inactivation and chelator stimulation of Streptococcus mitis arginine aminopeptidase

Activation of Streptococcus mitis (ATTC 9811) arginine aminopeptidase resulted in removal of the metal(s) from the enzyme molecule, and the action of the heavy metal ion in the inactivation process was shown to involve formation of a chelate complex between the enzyme molecule and metal or oxidation of functional group(s) on the enzyme surface. The enzyme also underwent activation by bovine serum albumin, amino acids, phosphate, and citric acid, which are probable physiological chelators.     

Metal ion binding and enzymatic mechanism of Methanococcus jannaschii RNase HII

RNase H degrades the RNA moiety in DNA:RNA hybrid in a divalent metal ion dependent manner. It is essential to understand the role of metal ion in enzymatic mechanism. One of the key points in this study is how many metal ions are involved in the enzyme catalysis. Accordingly, either one-metal binding mechanism or two-metal binding mechanism is proposed. We have studied the thermodynamic properties of four metal ions (Mg(2+), Mn(2+), Ca(2+), and Ba(2+)) binding to Methanococcus jannaschii RNase HII using isothermal titration calorimetry. All of the four metal ions were found to bind Mj RNase HII with 1:1 stoichiometry in the absence of substrate. Together with enzymatic activity assay data, we propose that only one metal ion binding to the enzyme in catalytic process. We also studied the pH dependence of metal binding and enzyme activity and found that at pH 6.5, Mg(2+) did not bind to the enzyme without the substrate but still activated the enzyme to about 2% of its maximum activity (in 10 mM Mn(2+) at pH 8). This implies that the substrate may also be incorporated in metal ion binding and help to position the metal ion. To find which acidic residues correspond to metal ion binding, we also studied the binding thermodynamics and enzymatic activity assay of four mutants: D7N, E8Q, D112N, and D149N in the presence of Mn(2+). The thermodynamic parameters are least affected for the D149N mutant, which has a very low enzymatic activity. This indicates that Asp149 is essential for the enzymatic activity. On the basis of all these observations, we suggest a metal binding model in which D7, E8, and D112 bind the metal ion and D149 activates a water molecule to attack the P-O bond in the RNA chain of the substrate.     

Human α‐amino‐β‐carboxymuconate‐ε‐semialdehyde decarboxylase (ACMSD): A structural and mechanistic unveiling

Human α‐amino‐β‐carboxymuconate‐ε‐semialdehyde decarboxylase determines the fate of tryptophan metabolites in the kynurenine pathway by controlling the quinolinate levels for de novo nicotinamide adenine dinucleotide biosynthesis. The unstable nature of its substrate has made gaining insight into its reaction mechanism difficult. Our electron paramagnetic resonance (EPR) spectroscopic study on the Cu‐substituted human enzyme suggests that the native substrate does not directly ligate to the metal ion. Substrate binding did not result in a change of either the hyperfine structure or the super‐hyperfine structure of the EPR spectrum. We also determined the crystal structure of the human enzyme in its native catalytically active state (at 1.99 Å resolution), a substrate analogue‐bound form (2.50 Å resolution), and a selected active site mutant form with one of the putative substrate binding residues altered (2.32 Å resolution). These structures illustrate that each asymmetric unit contains three pairs of dimers. Consistent with the EPR findings, the ligand‐bound complex structure shows that the substrate analogue does not directly coordinate to the metal ion but is bound to the active site by two arginine residues through noncovalent interactions. Proteins 2015; 83:178–187. © 2014 Wiley Periodicals, Inc.     

Muscle contraction: viscous-like frictional forces and the impulsive model

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.     

Calcium ion activation of rabbit liver alpha 1,2-mannosidase

Rabbit liver alpha 1,2-mannosidase is a calcium ion requiring enzyme involved in processing the asparagine-linked oligosaccharides of glycoproteins. Ca2+ activation occurs with an apparent Ka of 1.1 microM. The major effect of the metal ion activator is on Km rather than Vmax. The kinetic mechanism of the enzyme is that of an ordered equilibrium in which Ca2+ must bind before substrate and the metal ion cannot release once the substrate has added to the enzyme. Several other divalent cations including Co2+, Mn2+, and Zn2+ were competitive with Ca2+ and inhibited the enzyme. Significantly, Mg2+ had no effect on enzyme activity. 1-Deoxymannojirimycin and Tris, which inhibit glycoprotein processing in vivo, are inhibitors of the mannosidase competitive with substrate. The effect of Ca2+ on the affinity of the enzyme for substrate may be a determinant in regulation of enzyme activity in vivo.     

X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases

The alkaline phosphatase (AP) is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP). The crystal structure reveals many differences from other APs: 1) the catalytic residue is a threonine instead of serine, 2) there is no third metal ion binding pocket, and 3) the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily.     

Purification and kinetic mechanism of S-adenosylmethionine: myelin basic protein methyltransferase from bovine brain.

The enzyme S-adenosylmethionine (AdoMet): myelin basic protein (MBP) methyltransferase was purified 250-fold from bovine brain with an overall yield of 130%, relative to crude supernatant. The purification involves acid-base and (NH4)2SO4 precipitation, chromatography over Sephadex G-100 and DEAE-cellulose, followed by preparative isoelectric focusing. The enzyme has a pI of 5.60 +/- 0.05, and the Mr is estimated to be between 71,000 (from SDS/polyacrylamide-gel electrophoresis) and 74,500 (from gel filtration). The enzyme is stable at 37 degrees C for over 2 h, is stable frozen and does not require metal ions or reductants. The enzyme shows a high specificity for MBP and does not accept polyarginine as a substrate; F1 histone is methylated at 37% of the rate of MBP. Methylation occurs on an arginine residue in a single h.p.l.c.-resolvable peptide from the tryptic cleavage of MBP. Simple saturation kinetics are observed with respect to both substrates, with Km values of 18 microM and 32 microM for MBP and AdoMet respectively. The simplest kinetic mechanism that is consistent with the data requires ordered rapid-equilibrium binding, with AdoMet as the first substrate. The enzyme isolated in this work is different, both physically and kinetically, from the histone-specific arginine methyltransferases described by other workers. A new, simple, assay system for the methylation of MBP is described.     

Site-directed mutagenesis of mouse thymidylate synthase: alteration of Arg44 to Val44 in a conserved loop guarding the active site has striking effects on catalysis and nucleotide binding

The arginine located at position 44 of mouse thymidylate synthase is in a highly conserved loop that is in close proximity to the active site cleft of the enzyme. Structural analyses have suggested that this arginine forms hydrogen bonds with the alpha-carboxylate of the C-terminal amino acid and the phosphate of the substrate analog, FdUMP (D. A. Matthews, K. Appelt and S. J. Oatley, (1989) Adv. Enz. Reg., 29, 47-60). We have used protein engineering techniques to change this amino acid residue to valine. This alteration leads to large reductions in the ability of the enzyme to form covalent complexes with substrate (dUMP) or inhibitor (FdUMP) and at least a 100-fold reduction in catalytic activity. These observations show that this arginine plays an important role in maintaining catalytic activity of the enzyme.     

Mechanism of hydrolysis of phosphate esters by the dimetal center of 5'-nucleotidase based on crystal structures

5'-Nucleotidase belongs to a large superfamily of distantly related dinuclear metallophosphatases including the Ser/Thr protein phosphatases and purple acid phosphatases. The protein undergoes a 96 degrees domain rotation between an open (inactive) and a closed (active) enzyme form. Complex structures of the closed form with the products adenosine and phosphate, and with the substrate analogue inhibitor alpha,beta-methylene ADP, have been determined at 2.1 A and 1.85 A resolution, respectively. In addition, a complex of the open form of 5'-nucleotidase with ATP was analyzed at a resolution of 1.7 A. These structures show that the adenosine group binds to a specific binding pocket of the C-terminal domain. The adenine ring is stacked between Phe429 and Phe498. The N-terminal domain provides the ligands to the dimetal cluster and the conserved His117, which together form the catalytic core structure. However, the three C-terminal arginine residues 375, 379 and 410, which are involved in substrate binding, may also play a role in transition-state stabilization. The beta-phosphate group of the inhibitor is terminally coordinated to the site 2 metal ion. The site 1 metal ion coordinates a water molecule which is in an ideal position for a nucleophilic attack on the phosphorus atom, assuming an in-line mechanism of phosphoryl transfer. Another water molecule bridges the two metal ions.     

The effect of metal ions on the binding of ethanol to human alcohol dehydrogenase β<Subscript>2</Subscript>β<Subscript>2</Subscript>

Molecular docking simulations were performed in this study to investigate the importance of both structural and catalytic zinc ions in the human alcohol dehydrogenase beta(2)beta(2) on substrate binding. The structural zinc ion is not only important in maintaining the structural integrity of the enzyme, but also plays an important role in determining substrate binding. The replacement of the catalytic zinc ion or both catalytic and structural zinc ions with Cu(2+) results in better substrate binding affinity than with the wild-type enzyme. The width of the bottleneck formed by L116 and V294 in the substrate binding pocket plays an important role for substrate entrance. In addition, unfavorable contacts between the substrate and T48 and F93 prevent the substrate from moving too close to the metal ion. The optimal binding position occurs between 1.9 and 2.4 A from the catalytic metal ion.     

Regulatory role of polyamine in the acid phosphatase from potato tubers.

Effects of polyamine and metal ions on the new type of acid phosphatase purified from potato (Solanum tuberosum L. Irish Cobbler) tubers were analyzed. The enzyme belongs to nonspecific acid phosphatase family (EC 3.1.3.2), which hydrolyzes various phosphorylated substrates. The enzyme hydrolyzed inorganic pyrophosphate as a preferred substrate, and exhibited the hyperbolic kinetics with respect to the substrate, inorganic pyrophosphate in the absence of metal cations. Polyamine activated the enzyme effectively by lowering the K(m) value without appreciable changes in the maximal velocity. The most effective polyamines as activators were spermine and spermidine. Mg(2+) ion increased the K(m) value without affecting the maximal velocity of the enzyme, but Ca(2+) ion decreased both the K(m) and V(max) values. Increasing concentrations of spermine also decreased the K(m) value irrespective of Mg(2+) ion included, but gave a constant K(m) and V(max) values in the absence and presence of Ca(2+) ion. Action of spermine and metal ions can be explained by the complex formation with the substrate pyrophosphate. The acid phosphatase from potato can utilize the pyrophosphate-spermine or pyrophosphate-Ca(2+) complex as the preferred substrates. However, the enzyme can use the pyrophosphate-Mg complex with a weak affinity for the active site. Polyamine activates acid phosphatase in the absence and presence of metal cations, and activation by polyamine of the enzyme may contribute to the stimulation of starch biosynthesis and the control of glycolysis/gluconeogenesis by regulating PPi levels in growing potato tubers.     

The isolation and characterization of copper-resistant mutants ofAspergillus nidulans

The presence of aminopeptidases in the cytoplasm, in the cell wall, and in the cytoplasmic membrane fractions ofStreptococcus sanguis 903 was demonstrated by isoelectric focusing in combination with enzyme-staining procedures. The cytoplasm and the cell wall both had two aminopeptidases (pI 4.25 and 4.3) with broad substrate specificities and one enzyme (pI 4.2) specific for arginine substrates. The former enzymes were both stimulated by Co²⁺ ions; the latter enzyme had no metal cofactor. The cytoplasmic membrane aminopeptidase (pI 4.65) was arginine specific and was not stimulated by metal ions.     

The effect of divalent cations on the mode of action of DNase I. The initial reaction products produced from covalently closed circular DNA

The effect of different divalent metal ions on the hydrolysis of DNA by DNase I was studied with an assay which distinguishes between cleavage of one or both strands of the DNA substrate during initial encounters between enzyme and DNA. Using covalently closed superhelical SV40(I) DNA as substrate, initial reaction products consisting of relaxed circles or unit-length linears are resolved by electrophoresis of radioactively labeled DNA in agarose gels. Only in the presence of a transition metal ion, such as Mn2+ or Co2+, and only under certain reaction conditions, is DNase I able to cut both DNA strands at or near the same point, generating unit-length linears. This ability to cut both DNA strands is inhibited by such factors as temperature decrease, the addition of a monovalent ion or another divalent cation which is not a transition metal ion, or a reduction in the number of superhelical turns in the DNA substrate. All of these factors lead to a winding of the duplex helix and antagonize the unwinding of the duplex promoted by transition metal ion binding. Transition metal ions may thus convert the DNA substrate locally to a form in which DNase I can introduce breaks into both strands. In the presence of Mg2+, DNase I introduces single strand nicks into SV40(I), generating exclusively the covalently open, relaxed circular SV40(II) as the initial product of the reaction. In the presence of Mn2+, DNase I generates as initial products a mixture of SV40(II) and unit-length SV40 linear DNA molecules, formed by two nicks in opposite strands at or near the same point in the duplex. These circular SV40(II) molecules consist of two types. A minority class is indistinguishable from the nicked SV40(II) produced by DNase I in the presence of Mg2+. The majority class consists of molecules containing a gap in one of the two strands, the mean length of the gap being 11 nucleotides. The SV40(L) molecules produced in the presence of Mn2+ appear to have single strand extensions at one or both ends.     

Promiscuity comes at a price: Catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) is a highly promiscuous dinuclear metallohydrolase with respect to both substrate specificity and metal ion composition. While this promiscuity may adversely affect the enzyme's catalytic efficiency its ability to hydrolyse some organophosphates (OPs) and by-products of OP degradation have turned GpdQ into a promising candidate for bioremedial applications. Here, we investigated both metal ion binding and the effect of the metal ion composition on catalysis. The prevalent in vivo metal ion composition for GpdQ is proposed to be of the type Fe(II)Zn(II), a reflection of natural abundance rather than catalytic optimisation. The Fe(II) appears to have lower binding affinity than other divalent metal ions, and the catalytic efficiency of this mixed metal center is considerably smaller than that of Mn(II), Co(II) or Cd(II)-containing derivatives of GpdQ. Interestingly, metal ion replacements do not only affect catalytic efficiency but also the optimal pH range for the reaction, suggesting that different metal ion combinations may employ different mechanistic strategies. These metal ion-triggered modulations are likely to be mediated via an extensive hydrogen bond network that links the two metal ion binding sites via residues in the substrate binding pocket. The observed functional diversity may be the cause for the modest catalytic efficiency of wild-type GpdQ but may also be essential to enable the enzyme to evolve rapidly to alter substrate specificity and enhance k_cat values, as has recently been demonstrated in a directed evolution experiment. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.     

Transition metals as protease inhibitors

An alternative approach to the development of clinically useful protease inhibitors was investigated. The approach utilized coordination chemistry of transition metal ions rather than substrate analogs to block active sites of these enzymes. In the case of serine proteases it was found that aqueous Ti(IV) is a potent inhibitor of the trypsin subclass, but not the chymotrypsin subclass. The direct binding of Ti(IV) to trypsin was made possible by the presence of a free carboxyl group at the bottom of the substrate binding pocket of the enzyme, and the five-coordinate geometry of TiO(SO₄)(H₂O). Although initial binding of Ti(IV) was reversible, it was followed in time by irreversible inhibition. Direct binding of octahedral or tetrahedral metal ion complexes was prevented by the inability of the enzyme active sites to promote formation of a five-coordinate transition state of the metal ion required for reaction. These studies demonstrate the ability of direct metal ion binding as a way to enhance blocking of enzyme active sites as compared with that of traditional organic inhibitors. Application of these findings was investigated by measuring the affect Ti(IV) had on growth ofEscherichia coli, Salmonella typhimurium, andPseudotnonas aeruginosa. Five-coordinate titanyl sulfate completely inhibited the growth of these organisms. This suggests that five-coordinate titanyl sulfate, which is easier and less expensive to manufacture than conventional antibiotics, may be useful in controlling endemic infections ofE. coli andS. typhimurium.     

Mechanistic characterization of a bacterial malonate semialdehyde decarboxylase: identification of a new activity on the tautomerase superfamily

Malonate semialdehyde decarboxylase (MSAD) has been identified as the protein encoded by the orf130 gene from Pseudomonas pavonaceae 170 on the basis of the genomic context of the gene as well as its ability to catalyze the decarboxylation of malonate semialdehyde to generate acetaldehyde. The enzyme is found in a degradative pathway for the xenobiotic nematocide trans-1,3-dichloropropene. MSAD has no sequence homology to previously characterized decarboxylases, but the presence of a conserved motif (Pro1-(X)8 -Gly-Arg11-X-Asp-X-Gln) in its N-terminal region suggested a relationship to the tautomerase superfamily. Sequence analysis identified Pro1 and Arg75 as potential active site residues that might be involved in the MSAD activity. The results of site-directed mutagenesis experiments confirmed the importance of these residues to activity and provided further evidence to implicate MSAD as a new member of the tautomerase superfamily. MSAD is the first identified decarboxylase in the superfamily and is possibly the first characterized member of a new and distinct family within this superfamily. Malonate semialdehyde is analogous to a beta-keto acid, and enzymes that catalyze the decarboxylation of these acids generally utilize metal ion catalysis, a Schiff base intermediate, or polarization of the carbonyl group by hydrogen bonding and/or electrostatic interactions. A mechanistic analysis shows that the rate of the reaction is not affected by the presence of a metal ion or EDTA while the incubation of MSAD with the substrate in the presence of sodium cyanoborohydride results in the irreversible inactivation of the enzyme. The site of modification is Pro1. These observations are consistent with the latter two mechanisms, but do not exclude the first mechanism. Based on the sequence analysis, the outcome of the mutagenesis and mechanistic experiments, and the roles determined for Pro1 and the conserved arginine in all tautomerase superfamily members characterized thus far, two mechanistic scenarios are proposed for the MSAD-catalyzed reaction in which Pro1 and Arg75 play prominent roles.     

31P nuclear magnetic resonance spectroscopy studies of substrate and product binding to fructose-1,6-bisphosphatase.

The enzymatic hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi), which is catalyzed by fructose-1,6-bisphosphatase, has been studied by 31P nuclear magnetic resonance spectroscopy (NMR). At pH 7.5 and 15 degrees C, the equilibrium constant for the central complex K'eq = [E.Fru-6-P.Pi]/[E.Fru-1,6-P2.H2O] is about 2. This observation is in harmony with results obtained with a number of Bi Bi enzyme systems for the determination of K'eq in which a variety of experimental techniques were used (Knowles, J.R. (1980) Annu. Rev. Biochem. 49, 877-919). Significant changes in 31P NMR chemical shifts were observed for both the substrate, Fru-1,6-P2, and the product, Fru-6-P, when bound to the enzyme relative to ligand free in solution. The chemical shifts of the substrate and product were altered further in the presence of Mg2+, the catalytic divalent metal ion. The chemical shifts caused by the addition of metal ion can be reversed in the presence of trans-1,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid (CDTA) or AMP. In the presence of the metal ion chelator or the nucleotide, the substrate had a chemical shift that was about the same as that observed in the absence of metal ion. On the basis of these observations we suggest that AMP and CDTA exhibit similar effects, i.e. they both remove the catalytic metal ion from the enzyme. This finding is supportive of the suggestion (Scheffler, J. E., and Fromm, H.J. (1986) Biochemistry 25, 6659-6665; Liu, F., and Fromm, H.J. (1990) J. Biol. Chem. 265, 7401-7406) that the role of AMP in the regulation of fructose-1,6-bisphosphatase is to prevent binding of the divalent metal activator to the enzyme.     

Cyanide formation from histidine in Chlorella. A general reaction of aromatic amino acids catalyzed by amino acid oxidase systems.

The formation of HCN from D-histidine in Chlorella vulgaris extracts is shown to be due to the combined action of a soluble protein and a particulate component. Either horse-radish peroxidase (EC 1.11.1.7) or a metal ion with redox properties can be substituted for the particulate component. Ions of manganese and vanadium are especially effective, as are o-phenanthroline complexes of iron. Cobalt ions are less active. The D-amino acid oxidase (EC 1.4.3.3) from kidney and the L-amino acid oxidase (EC 1.4.3.2) from snake venom likewise cause HCN production from histidine when supplemented with the particulate preparation from Chlorella or with peroxidase or with a redox metal ion. The stereospecificity of the amino acid oxidase determines which of the two stereoisomers of histidine is active as an HCN precursor. Though histidine is the best substrate for HCN production, other naturally occurring aromatic amino acids (viz. tyrosine, phenylalanine and tryptophan) can also serve as HCN precursors with these enzyme systems. The relative effectiveness of each substrate varies with the amino acid oxidase enzyme and with the supplement. With respect to this latter property, the particulate preparation from Chlorella behaves more like a metal ion than like peroxidase.