The pH dependence of peptide hydrolysis by nitrocarboxypeptidase A

Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K_m is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pK_E1 of 6.37 ± 0.19 and pK_E2 of 6.60 ± 0.17 in $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, and apparent pK of 5.59 ± 0.06 in K_cat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK_E2 in the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ profile, which appears to be shifted from a higher value, pK_E2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k_cat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.     

Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.     

A steady-state kinetic study of the ribonuclease A catalyzed hydrolysis of uridine-2′:3′(cyclic)-5′-diphosphate

Initial velocity measurements were made on the ribonuclease A catalyzed hydrolysis of P-5′-Urd-2′:3′-P in the pH range 4.0–8.0 at 25 °C in 0.1 m Tris-acetate/0.1 m KCl. The pH dependence of the Michaelis constant, K_m, the turnover number k_s, and $\text{k}{}_{\mathrm{s}}\text{K}{}_{\mathrm{m}}$ for P-5′-Urd-2′:3′-P were similar to those reported for Urd-2′:3′-P (5). When P-5′-Urd-2,3-P and Urd-2′:3′-P were compared under similar conditions the average difference in k_s and K_m indicated that these parameters were 5-fold and 23-fold lower, respectively, for P-5′-Urd-2′:3′-P. The slight difference in the pH dependence of $\text{k}{}_{\mathrm{s}}\text{K}{}_{\mathrm{m}}$ for these two substrates can be interpreted in terms of a specific interaction of the enzyme at the 5′ position of P-5′-Urd-2′:3′-P, which permits a less exclusive dependence on the ionized state of the free enzyme in binding this substrate. The nature of the interaction of the substrate 5′-phosphomonoester group with the enzyme is discussed in terms of possible interactions with Lys-41 and His-119.     

Hymenolepis diminuta: The non-saturable component of methionine uptake

Lussier P. E., Podesta R. B. and Mettrick D. F. 1982. Hymenolepis diminuta: the non-saturable component of methionine uptake. International Journal for Parasitoiogy12: 265–270. The concentration dependence of in vitro unidirectional methionine influx by Hymenolepis diminuta was analysed by the relation: $\text{J =}\text{(J}{}_{\mathrm{<mtext>m</mtext>}}\text{C}{}_{\mathrm{<mtext>b</mtext>}}\text{)}\text{(K}{}_{\mathrm{t}}\text{+ C}{}_{\mathrm{<mtext>b</mtext>}}\text{)}\text{+ K}{}_{\mathrm{d}}\text{(C}{}_{\mathrm{<mtext>b</mtext>}}\text{)}$, where J_m is the maximum uptake rate, K_t is the the apparent affinity constant and C_b is the medium substrate concentration. The linear component was separated using an asymptotic least squares curve fitting procedure and the resulting constant, K_d, is thought to be an apparent permeability coefficient. K_d may be a reflection of a simple diffusive component, a second mediated component or a combination of a passive and mediated influx. The low Q₁₀ value of the K_d's for methionine uptake (Q₁₀ = 1.31) indicated that this component is probably a reflection of diffusion within the membrane. However, the decrease in the K_d component in the presence of leucine and glycine, implies that there is also a small, second, mediated component in addition to the diffusive component. K_d derived from the asymptotic portion of the concentration-flux relation was compared with the residual flux of methionine after near complete inhibition of the mediated component with leucine and glycine. The K_d component was found to be pH-sensitive, increasing as the pH decreased and was not affected by external sodium. Results indicate that the mediated component of methionine influx was accelerated by increasing external Na⁺ and H⁺ concentrations.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase

Quercetin inhibited a dog kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ preparation without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP or $\text{K}{}_{0.5}$ for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP and the $\text{K}{}_{0.5}$ for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for substrate, and $\text{K}{}_{0.5}$ for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations.     

Equilibrium constants for association of guanidinium and ammonium ions with oxyanions: The effect of changing basicity of the oxyanion

Using guanidinium and n-butylammonium cations (C⁺) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H₂A, three association complexes may exist: $\text{K}{}_{\mathrm{<mtext>1M</mtext>}}\text{=}\text{[CHA]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[HA}{}^{\mathrm{?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>1D</mtext>}}\text{=}\text{[CA}{}^{\mathrm{?}}\text{]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[A}{}^{\mathrm{2?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>2D</mtext>}}\text{=}\text{[C}{}_2\text{A]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[CA}{}^{\mathrm{?}}\text{]}$. For guanidinium ion and phosphate, K_1M = 1.4, K_1D = 2.6, and K_2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pK_a = 4.8, K_1M = 0.37; formate, pK_a = 3.8, K_1M = 0.32; and chloroacetate, pK_a = 2.9, K_1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.     

Bacillus subtilis aminopeptidase: specificity toward amino acyl-beta-naphthylamides.

The kinetic parameters for the hydrolyses of different l-α-amino acid-β-naphthylamides by Bacillus subtilis aminopeptidase have been measured for the native enzyme and for the enzyme activated in 5 mm Co(NO₃)₂. In most cases Co²⁺ activation decreased K_m(app) values and increased k_cat values, in other cases k_m(app) and k_cat values were increased; for the remainder of the substrates tested k_m(app) values and k_cat values were decreased. In all cases tested the ratios of $\text{(}\text{k}\text{cat}\text{K}{}_{\mathrm{<mtext>m(</mtext><mtext>app</mtext><mtext>)</mtext>}}\text{)}\text{CO}{}^{\mathrm{2+}}\text{/(}\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{<mtext>m(</mtext><mtext>app</mtext><mtext>)nativ</mtext>}}\text{)}$ were increased (2- to 108-fold). For the native enzyme the order of specificity toward the l-amino acid-β-naphthylamides was Arg > Met > Trp > Lys > Leu and for the Co²⁺ activated enzyme the order of specificity was Lys > Arg > Met > Trp > Leu. The native enzyme hydrolyzed Pro-β-naphthylamide, but not α-Glu-β-naphthylamide; Co²⁺ activation of the enzyme affected an appreciable rate of hydrolysis of the latter substrate.     

Graphic method for determination of allosteric constants

The maximum slope of the plot, appearing in the paper of Watari & Isogai (1976), was derived algebraically as a function of allosteric constants c and α_mor β_m (= cα_m), and the relation between L, c, and α_mor β_m, was also obtained, where $\text{L =}\text{T}{}_{\mathrm{o}}\text{R}{}_{\mathrm{o}}\text{, c =}\text{K}{}_{\mathrm{R}}\text{K}{}_{\mathrm{T}}\text{, α}{}_{\mathrm{m}}\text{=}\text{F}{}_{\mathrm{m}}\text{K}{}_{\mathrm{R}}\text{, β}{}_{\mathrm{m}}\text{=}\text{F}{}_{\mathrm{m}}\text{K}{}_{\mathrm{T}}\text{, R}{}_{\mathrm{o}}\text{and}\text{T}{}_{\mathrm{o}}$ are concentrations of unligated R and T states respectively, K_Rand K_T are microscopic dissociation constants, and F_m is the ligand concentration at the maximum slope of the plot. When the maximum slope is increased by one, the value becomes Hill constant, n. Nomographs which enable easier estimation of allosteric constants, L and c, were constructed from the two given values, the maximum slope of the plot, n ? 1, and α_mor β_m, in the cases where the maximum number of ligands, N, was 2 and 4. In the nomograph, log c is plotted against log L²c^N keeping the value of the maximum slope of the plot and that of α_mor β_m constant. These nomographs show that the representation is symmetrical in the cases of L²c^N > 1 and L²c^N < 1.     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

Alpha-chymotrypsin: effects of bicyclic substrate geometry and hydrophobicity on reactivity

The α-chymotrypsin-catalyzed hydrolysis rates of p-nitrophenyl cyclopentane-carboxylate (I), p-nitrophenyl indan-2-carboxylate (II), and p-nitrophenyl spiro-[4.4]nonane-2-carboxylate (III) were measured at pH 8.1 in 20% methanol. After correction for variations in reactivity owing to stereoelectronic effects inherent to the substrates, the deacylation rate constants (k_c)_n of I and II are not significantly different. In $\text{(}\text{k}{}_{\mathrm{c}}\text{K}{}_{\mathrm{m}}\text{)}{}_{\mathrm{n}}$ II is 50 times more reactive than I, which demonstrates that the aromatic ring of the former substrate contributes significantly to its reactivity. The nearly equal reactivities of II and III indicate that the enzyme is rather insensitive to the geometry of the nonester-bearing ring of these compounds.     

pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The V_max for the demethylation reaction exhibited a pH optimum at approximately 4.5. The K_m for N,N-dimethylaniline increased with decreasing pH, while the K_m for ethyl hydroperoxide varied in a manner paralleling V_max. Comparison of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the V_max versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.     

Isolation and characterization of isocitrate lyase of castor endosperm

Isocitrate lyase (threo-D_S-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A₀ and A_t are activities at time zero at t, and k₁ and k₂ are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pK_a equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg²⁺ (5.0 mm) to a solution of this protein. Addition of Ba²⁺ or Mn²⁺ brings about partial recovery. Other metal ions are not effective.     

Structure-activity relationships in papain and bromelain ligand interactions

The parameters K_m and k_cat were determined for 16 methyl hippurates (CH₃OCOCH₂NHCOC₆H₄-X) hydrolyzed by papain. A simple linear relationship is found between log $\text{1}\text{K}{}_{\mathrm{m}}$ and the hydrophobic substituent constant π. It is found that log k_cat is parabolically related to π. The results with papain are compared with results obtained by Hawkins and Williams with the enzyme bromelain. The two enzymes behave in a similar fashion.     

Kinetics and mechanism of the F1 isozyme of horse liver aldehyde dehydrogenase.

The reaction mechanism of the F1 isozyme of horse liver aldehyde dehydrogenase (EC 1.2.1.3) was investigated using both steady-state and rapid kinetic techniques. Using the steady-state substrate velocity patterns, the NADH inhibition patterns at several aldehyde concentrations, and the substrate analog (adenosine diphosphoribose and chloral hydrate) inhibition patterns, the enzymic catalysis was shown to involve ordered addition of NAD followed by aldehyde. This mechanism was confirmed using the kinetics of the hydrolysis of p-nitrophenyl acetate as an indicator of the dehydrogenase substrate binding. Steady-state experiments with deuteroacetaldehyde showed the V to be unchanged, but the K_m increased (). Stopped flow experiments where E-NAD was rapidly mixed with aldehyde showed a burst of NADH formation followed by slower steady-state turnover. This result clearly indicates that the rate limiting step lies after NAD reduction. The NADH off rate (0.7 s^?1) as estimated by displacement of NADH from the E-NADH complex upon rapid addition of NAD was found to be very close to the steady-state site turnover number (0.3 s^?1). This fact and the relatively small effect of aldehyde R-group on maximal velocity suggest that the slow rate of NADH release contributes significantly to limitation of the enzyme catalytic velocity.     

An improved spectrophotometric assay for human plasma carboxypeptidase N

A continuous spectrophotometric assay for human plasma carboxypeptidase N utilizing furylacryloyl-alanyl-lysine is described. Synthesis was made by use of 9-(2-sulfo)fluorenylmethyloxycarbonyl (Sulfmoc) chloride as the N-?-amino-blocking group for lysine. The substrate has the advantage of containing a chromophore which allows difference measurements above 324 nm. The kinetic parameters K_m and $\text{K}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ have been determined for furylacryloyl-alanyl-lysine and -arginine. Difference measurements were related to micromoles of lysine or arginine released and were expressed as units.     

Further studies on anthranilate N-acetyltransferase and the metabolism of N-acetylanthranilic acid in Aerobacter aerogenes

Antranilate N-acetlytransferase, which is a constitutive enzyme, is responsible for the formation of N-acetylanthranilic acid which accumulated int he culture medium of certain mutants of Aerobacter aerogenes. It has been shown to be dissimilar to serine O-acetyltrasferase and not to be involved in the acetylation of a variety of aliphatic compounds. Aniline and m-aminobenzoic acid are, however, readily acetylated, the K_m for the latter compound being the same as that for anthranilic acid, 13 mM. p-Aminobenzoic acid is only slowly acetylated and salicylic acid only acted as an inhibitor of the reaction. $\text{N-[}{}^3\text{H}\text{]}\text{Acetyl}\text{[1,7-}{}^{14}\text{C}{}_2\text{]}\text{anthranili}$ acid was prepared but could not be shown to be deacylated for further metabolized when administered to any whole cell, cell extract or toluene-lysed cell preparation.     

Malate dehydrogenase. Kinetic studies with meso-tartrate and 2-keto-3-hydroxysuccinate, comparison of the mitochondrial and supernatant pig heart enzymes.

Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10^?6, M^?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{V}{}_{\mathrm{<mtext>D</mtext>}}$ and $\text{(}\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{V}{}_{\mathrm{<mtext>D</mtext>}}\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme.     

Trichomonas vaginalis: NADH oxidase activity.

NADH oxidase activity was detected in the 105,000g supernatant (“soluble”) fraction of Trichomonas vaginalis and the enzyme was purified 50-fold by centrifugation, ammonium sulfate precipitation, Sephadex G-200, and DEAE-Sephadex A-25 chromatography. The ratio of oxygen uptake to NADH oxidation was approximately one-half. Addition of catalase did not affect the rate of oxygen uptake elicited by NADH. Since the purified fraction was free from interfering enzymes, the postulated reaction is as follows: $\text{NADH}\text{+}\text{H}{}^{\mathrm{+}}\text{+}\text{1}\text{2}\text{= NAD}{}^{\mathrm{+}}\text{+ H}{}_2\text{O}$. Among numerous substances tested, only NADH was a functional substrate, whereas NADPH was not oxidized. The purified enzyme had a V_max of 16.5 μmole of oxygen consumed/min/mg protein, and the apparent K_m for NADH was 7.4 μM. Substrate inhibition was observed at 3.7 mM NADH. The purified NADH oxidase was competitively inhibited by NAD⁺ as well as by NADP⁺ with 50% inhibition at 1 and 5 mM, respectively. The enzyme was also markedly inhibited by p-chloromercuribenzoate, hydrogen peroxide, and transient metal-chelators such as bathophenanthroline or o-phenanthroline. A flavoprotein antagonist, atebrin was slightly less inhibitory. Various quinones, flavin nucleotides and artificial dyes, except for p-benzoquinone, ferricyanide and cytochrome c, did not function in accepting electrons from NADH oxidase. These three compounds, however, were still poor electron acceptors in the enzymatic reaction suggesting that the trichomonad NADH oxidase has little diaphorase activity. All of these findings indicate that T. vaginalis has an unique NADH oxidizing enzyme in that H₂O seems to be the prdouct of oxygen reduction. This NADH oxidase appears important in the aerobic metabolism of this parasite.