Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase.

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7--0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

Purification and properties of a pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15

A pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was purified to homogeneity. The enzyme is a tetramer (Mr 170,000) of identical subunits and binds 4 pyridoxal-P/tetramer; it is resolved by dialysis against cysteine at pH 6.8. Between pH 6.2 and 8.8, the holoenzyme shows pH-independent absorbance maxima at 333 and 416 nm. Vmax/Km is highest at pH 6.5; this optimum reflects chiefly increased Km values for histidine at lower or higher pH values, whereas Vmax is highest at pH 5.0 and decreases only moderately between pH 5.0 and 8.0. The enzyme also decarboxylates beta-(2-pyridyl)alanine and N tau-methylhistidine (but not N pi-methylhistidine); arginine, lysine, and ornithine are neither substrates nor inhibitors. The hydrazine analogue of histidine, 2-hydrazino-3-(4-imidazolyl)propionic acid, is a very potent competitive inhibitor; other carbonyl reagents and a variety of carboxyl- or amino-substituted histidines also inhibit competitively. alpha-Fluoromethylhistidine is a potent irreversible inhibitor of the enzyme; alpha-methylhistidine is a competitive inhibitor/substrate that is decarboxylated slowly and undergoes a slow decarboxylation-dependent transamination that converts the holoenzyme to pyridoxamine-P and apoenzyme. Dithiothreitol and other simple thiols are mixed-type inhibitors that interact with pyridoxal-P at the active site to form complexes (lambda max congruent to 340 nm), presumably the corresponding thioalkylamines, without resolving the holoenzyme. This histidine decarboxylase (Vmax = 72 mumol X min-1 X mg-1) is much more active than "homogeneous" preparations of mammalian pyridoxal-P-dependent histidine decarboxylase (Vmax congruent to 1.0) and is about equal in activity to the pyruvoyl-dependent histidine decarboxylases from Gram-positive bacteria.     

Assay of L-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.     

Purification of glutamate dehydrogenase from the rabbit liver and study of its major physico-chemical properties

Highly purified preparations of glutamate dehydrogenase were obtained from mitochondrial and cytoplasmic fractions of rabbit liver by affinity chromatography on CL-Sepharose 4B modified by adenosine diphosphate. Some physico-chemical properties of the purified enzymes (e. g., specific activity, molecular weight, quaternary structure, stability against denaturating effect of urea, pH optimum of catalyzed reactions, Km values for substrates and coenzymes) were found to be identical. The sole difference was detected in the ability of enzyme preparations to be activated by adenosine diphosphate. The activation of the cytoplasmic enzyme is 160%, that of mitochondrial glutamate dehydrogenase is 230-240% under the same conditions.     

In vitro effects of pH and phosphorylation on neostriatal tyrosine hydroxylase from control and haloperidol-treated rats.

Acute administration of haloperidol to rats causes a marked decrease in the Km of neostriatal tyrosine hydroxylase for the pteridine cofactor, 6MPH₄, with no change in Vmax. We report that this effect is dependent on the pH of the assay mixture. It occurs at pH 6.5 but not at pH 6.0, the pH optimum for TH. With phosphorylating conditions at pH 6.5, the haloperidol-induced activation is no longer observed and the kinetics of TH are the same as those from control rats. At pH values of 6.0, 6.3 and 6.5, a significant decrease in Vmax occurs, with increasing pH, while no significant change in Km for the cofactor is observed for TH from control rats. However, when phosphorylating conditions are employed a marked increase in Km for the cofactor is observed while only a slight decrease in Vmax is seen, with increasing pH, for the control enzyme at the three pH values tested.     

Dehydrogenation by gluconate 6-phosphate dehydrogenase of an isosteric phosphonate substrate analogue.

6,7-Dideoxy-D-gluco-heptonic-7-phosphonic acid, the isosteric phosphonate analogue of gluconate 6-phosphate, was prepared by incubation of the corresponding analogue of glucose 6-phosphate with glucose 6-phosphate dehydrogenase and NADP+ in the presence of an enzyme NADPH-NADP+ recycling system. The analogue of gluconate 6-phosphate is a substrate for yeast gluconate 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH 7.5 and 8.0. At both pH values the Km values are approx. 3-fold higher and the Vmax. values approx. 7-fold lower than those of the natural substrate.     

Effect of Free Malonate on the Utilization of Glutamate by Rat Brain Mitochondria

Malonate is an effective inhibitor of succinate dehydrogenase in preparations from brain and other organs. This property was reexamined in isolated rat brain mitochondria during incubation with L-glutamate. The biosynthesis of aspartate was determined by a standard spectrofluorometric method and a radiometric technique. The latter was suitable for aspartate assay after very brief incubations of mitochondria with glutamate. At a concentration of 1 mM or higher, malonate totally inhibited aspartate biosynthesis. At 0.2 mM, the inhibitory effect was still present. It is thus possible that the natural concentration of free malonate in adult rat brain of 192 nmol/g wet weight exerts an effect on citric acid cycle reactions in vivo. The inhibition of glutamate utilization by malonate was readily overcome by the addition of malate which provided oxaloacetate for the transamination of glutamate. The reaction was accompanied by the accumulation of 2-oxoglutarate. The metabolism of glutamate was also blocked by inclusion of arsenite and gamma-vinyl-gamma-aminobutyric acid but again added malate allowed transamination to resume. When arsenite and gamma-vinyl-gamma-aminobutyric acid were present, the role of malonate as an inhibitor of malate entry into the mitochondrial interior could be determined without considering the inhibition of succinate dehydrogenase. The apparent Km and Vmax values for uninhibited malate entry were 0.01 mM and 100 nmol/mg protein/min, respectively. Malonate was a competitive inhibitor of malate transport (Ki = 0.75 mM).     

Ammonia formation in isolated rat liver mitochondria

Studies of isolated rat liver mitochondria were undertaken in order to evaluate the importance of glutamate transport, oxidation reduction state, and product inhibition on the rates of formation of ammonia from glutamate. Uptake and efflux of glutamate across the mitochondrial membrane were measured isotopically in the presence of rotenone. Efflux was stimulated by H+ in the mitochondrial matrix and was found to be first order with respect to matrix glutamate except when the matrix pH was unphysiologically low. The data suggest that the Km of matrix glutamate for efflux is decreased by H+. Matrix H+ also appeared to stimulate glutamate uptake, but the effect was to increase both the Km of medium glutamates and Vmax. Mitochondria were incubated at 15 and 28 degrees C with glutamate and malonate. Under these conditions, glutamate was metabolized only by the deamination pathway. Flux was evaluated by assay of ammonia formation. Oxidation reduction state was varied with ADP and uncoupling agents. Matrix alpha-ketoglutarate was varied either by the omission of malonate from the incubation media or by adding alpha-ketoglutarate to the external media. Influx and efflux of glutamate could be calculated from previously determined transport parameters. The difference between calculated influx and efflux was found to be equal to ammonia formation under all conditions. It was, therefore, possible to evaluate the relative contributions of oxidation reduction state, transport, and product inhibition as effectors of ammonia formation. The contribution of transport was relatively small while oxidation reduction state exerted a large influence. alpha-Ketoglutarate was found to be a potent competitive inhibitor of ammonia production and glutamate dehydrogenase. Inhibition of glutamate dehydrogenase by alpha-ketoglutarate was judged to be a potentially important modulator of metabolic fluxes.     

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80-1000 microM) both Km and Vmax values were comparable. With the HPLC assay the concentration range was extended down to 12 microM and initial rates could be determined. The apparent Km was about 12 microM. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.     

Human liver folylpolyglutamate synthetase: biochemical characterization and interactions with folates and folate antagonists

Folylpolyglutamate synthetase (FPGS) was isolated from human liver cytosol by 0-30% (w/v) ammonium sulfate fractionation and characterized biochemically. Using aminopterin (AMT), L-[3H]glutamate and MgATP as cosubstrates, maximal gamma-L-glutamylation activity was observed in the presence of the activators KCl and NaHCO3. ATP and 2-mercaptoethanol were each required for enzyme activity and stability. In the absence of ATP, human liver FPGS rapidly inactivated at 37 degrees C (t1/2 approximately 8 min), whereas FPGS isolated from rabbit liver was significantly more stable (t1/2 = 68 min). Both folates and antifolates were effectively polyglutamylated by the isolated human liver enzyme. Km parameters determined for AMT (Km = 4.3 microM) were similar to those determined for several reduced folates (tetrahydrofolic acid, dihydrofolic acid, and folinic acid; Km = 3-7 microM), while significantly higher Km values were observed for methotrexate (MTX) and 5-methyltetrahydrofolic acid (Km = 50-60 microM) and for folic acid (Km = 100 microM). All of the substrates examined exhibited Vmax values ranging from 30 to 90% of the AMT value (Vmax = 935 pmol product/mg/h). The order of reactivity for these substrates differed from that determined in parallel studies for FPGS isolated from rat and rabbit liver. In the case of AMT and several reduced folates, inhibition of human liver FPGS was observed at substrate concentrations at or above 50-250 microM. FPGS isolated from six individual human livers exhibited highly similar biochemical and kinetic properties, suggesting the presence of the same or at least highly similar enzyme species in each individual, with a five-fold interindividual range in specific activities observed. Comparison of MTX with its higher polyglutamates (MTX-Glu2 to MTX-Glu6) as FPGS substrates indicated a significant decrease in Vmax values with increasing glutamate chain length which was partially compensated for by a corresponding decrease in Km. Consistent with these observations, the isolated enzyme was unable to synthesize polyglutamates higher than MTX-Glu3 when MTX was supplied as substrate, raising the question as to how MTX polyglutamates containing up to five or six gamma-L-glutamate residues are formed in vivo.     

Crystallization and partial characterization of glutamate dehydrogenase from ox liver nuclei.

Glutamate dehydrogenase have been obtained in crystalline form from purified ox liver nuclear fractions. The enzyme appeared homogeneous, as judged by several electrophoretic techniques at two pH values. A comparative study with the widely known ox liver mitochondrial glutamate dehydrogenase revealed several common features, such as the allosteric effect of the nucleotides ADP and GTP, the activation at high concentrations of the cofactor NAD+, and the existence of a concentration-dependent reversible monomer-polymer(s) equilibrium. However, the two enzymes differed in many other respects. Inorganic phosphate activated nuclear glutamate dehydrogenase to a much greater extent than the mitochondrial enzyme; the substrate NH4+ showed cooperative homotropic interactions only with nuclear glutamate dehydrogenase; kinetic differences were detected with most of the reaction substrates, as well as different rates of oxidative deamination of other L-amino acids, the nuclear enzyme had a higher anodic mobility and a different chromatographic behavior on anionic exchangers. The latter evidence indicates that the glutamate dehydrogenase activity in liver is associated with two proteins which are structurally different, thus confirming the results of a separate immunological study. Preliminary evidence suggests that the enzyme in nuclei is attached to the nuclear envelope, probably the inner membrane, from which it can be solubilized by the addition of salts.     

Effect of inorganic pyrophosphate and its diphosphonate analogs on glucose-6-phosphatase dehydrogenase activity of mouse organs

The glucose-6-phosphatase dehydrogenase (EC 1.1.1.49) reaction of mouse organs was studied as affected by PPi and its diphosphonate analogs. It is shown that in vitro and hydroxy-1-ethane-1,1-diphosphonic acid) inhibit the mentioned enzyme of the mouse spleen and liver. The effect of hydroxyl-1-ethane-1,1-diphosphonic acid was used as an example to show that inhibition of glucose-6-phosphate dehydrogeanse by diphosphonates belongs to the mixed type characterized by changes in the Km and Vmax values. For the spleen enzyme Km equals 0.064 mM, Vmax - 4.7 Mg of NADPH per 1 mg of protein-1. h-1. Administration of methylene diphosphonic acid causes an inhibition in vivo of the glucose-6-phosphatase dehydrogenate activity of the liver but not of the spleen and thymus. Basing on the isoenzymic composition of the enzyme for the mentioned organs, it is possible to suppose that the difference in the methylene diphosphonic acid effect in the liver and lymphoid organs may depend on the differences in its isoenzymic spectrum. The fact that in vivo methylene diphosphonic acid in a dose having an immuno-depressive action has no influence on the activity of glucose-6-phosphatase dehydrogenase in the lymphoid organs, may evidence for the absence of the indirect immunodepressive effect of diphosphonate by affecting this enzyme.     

Inhibition of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide

Renal hyperosmotic conditions may produce reactive oxygen species, which could have a deleterious effect on the enzymes involved in osmoregulation. Hydrogen peroxide was used to provoke oxidative stress in the environment of betaine aldehyde dehydrogenase in vitro. Enzyme activity was reduced as hydrogen peroxide concentration was increased. Over 50% of the enzyme activity was lost at 100 μM hydrogen peroxide at two temperatures tested. At pH 8.0, under physiological ionic strength conditions, peroxide inhibited the enzyme. Initial velocity assays of betaine aldehyde dehydrogenase in the presence of hydrogen peroxide (0-200 μM) showed noncompetitive inhibition with respect to NAD(+) or to betaine aldehyde at saturating concentrations of the other substrate at pH 7.0 or 8.0. Inhibition data showed that apparent V(max) decreased 40% and 26% under betaine aldehyde and NAD(+) saturating concentrations at pH 8.0, while at pH 7.0 V(max) decreased 40% and 29% at betaine aldehyde and NAD(+) saturating concentrations. There was little change in apparent Km(NAD) at either pH, while Km(BA) increased at pH 7.0. K(i) values at pH 8 and 7 were calculated. Our results suggest that porcine kidney betaine aldehyde dehydrogenase could be inhibited by hydrogen peroxide in vivo, thus compromising the synthesis of glycine betaine.     

Evidence for an essential histidine in neutral endopeptidase 24.11

Rat kidney neutral endopeptidase 24.11, "enkephalinase", was rapidly inactivated by diethyl pyrocarbonate under mildly acidic conditions. The pH dependence of inactivation revealed the modification of an essential residue with a pKa of 6.1. The reaction of the unprotonated group with diethyl pyrocarbonate exhibited a second-order rate constant of 11.6 M-1 s-1 and was accompanied by an increase in absorbance at 240 nm. Treatment of the inactivated enzyme with 50 mM hydroxylamine completely restored enzyme activity. These findings indicate histidine modification by diethyl pyrocarbonate. Comparison of the rate of inactivation with the increase in absorbance at 240 nm revealed a single histidine residue essential for catalysis. The presence of this histidine at the active site was indicated by (a) the protection of enzyme from inactivation provided by substrate and (b) the protection by the specific inhibitor phosphoramidon of one histidine residue from modification as determined spectrally. The dependence of the kinetic parameter Vmax/Km upon pH revealed two essential residues with pKa values of 5.9 and 7.3. It is proposed that the residue having a kinetic pKa of 5.9 is the histidine modified by diethyl pyrocarbonate and that this residue participates in general acid/base catalysis during substrate hydrolysis by neutral endopeptidase 24.11.     

Structural role of histidine residues in NAD(P)-glutamate dehydrogenase from the bovine liver

Photooxidation of bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3) in the presence of methylene blue at a low light intensity occurs in two stages. At the first stage, the duration of which depends on temperature and dye concentration, a slight activation is observed simultaneously with the oxidation of two histidine residues. At the second stage, the inactivation is concomitant with the oxidation of three histidine and one tryptophan residues. The inactivation is a first order reaction (k = 3,22 X 10(-2) min-1) and is correlated with changes in the circular dichroism spectra. These data testify to the structural role of histidine residues in the GDH molecule. The kinetic behaviour of GDH during its modification with diethylpyrocarbonate (DEP) depends on pH and the reagent concentration. Four histidine residues undergo carbethoxylation at pH 6.0 and 7.5, but the modification rate is much higher at pH 7.5. At low DEP concentrations, a remarkable activation is observed with a simultaneous modification of one histidine residue, which is independent of pH. At high DEP concentrations, a rapid inactivation takes place at pH 7.5. Treatment of the carbethoxylated inactive enzyme with hydroxylamine results in the deacylation of histidine residues without any noticeable reactivation. The data on the combined effect of DEP and pyridoxal-5'-phosphate suggest that GDH inactivation by DEP at pH 7.5 is a result of modification of an essential epsilon-NH2 group of lysine-126.     

Evidence for a functional role for histidine in lysyl oxidase catalysis

The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.     

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in canine pancreas

Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with steroid-delta 5-4-isomerase was demonstrated for the first time in the pancreas. The enzyme complex was assayed by measuring the conversion of pregnenolone to progesterone as well as of dehydroepiandrosterone to androstenedione and found to be localized primarily in the mitochondrial fraction of dog pancreas homogenates. The delta 5-3 beta-hydroxysteroid dehydrogenase used either NAD+ or NADP+ as co-substrates, although maximal activity was observed with NAD+. In phosphate buffer, pH 7.0 and 37 degrees C, the apparent Km values of the dehydrogenase were 6.54 +/- 0.7 microM for pregnenolone and 9.61 +/- 0.8 microM for NAD+. The apparent Vmax was determined as 0.82 +/- 0.02 nmol min-1 mg-1. Under the same conditions the Km values for dehydroepiandrosterone and NAD+ were 3.3 +/- 0.2 microM and 9.63 +/- 1.6 microM, respectively, and the apparent Vmax was 0.62 +/- 0.01 nmol min-1 mg-1.     

Purification of 2-oxoaldehyde dehydrogenase and its dependence on unusual amines.

1. 2-Oxoaldehyde dehydrogenase was purified from sheep liver and gave one band on polyacrylamide-gel electrophoresis. 2. The enzyme was completely dependent for its activity on the presence of Tris or one of a number of related amines, all of general structure: (See article). When more than one R group was hydrogen no enzyme activity was observed. 3. Only one of these amines is known to exist in living tissues and large concentrations of all amines were required for maximum activity. L-2-Aminopropan-1-ol was the most effective amine on the basis of substrate Km and Vmax. values and the amine Km values. 4. The enzyme was activated by phosphate which lowered the Km values for methylglyoxal, amine and NAD+. 5. The pH optimum of the enzyme was 9.3 and there was no activity at pH values below 7.8. A search for activators that might produce activity at pH 7.4 proved unsuccessful. 6. The enzyme was inhibited by rather large concentrations of barbiturates (6-46 mM) and nitro-alcohol analogues of the activating amines (66-139 mM).     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.