Mechanism of reactions catalyzed by selenocysteine beta-lyase

The reaction mechanism of selenocystine beta-lyase has been studied and it was found that elemental selenium is released enzymatically from selenocysteine, and reduced to H2Se nonenzymatically with dithiothreitol or some other reductants that are added to prepare selenocysteine from selenocystine in the anaerobic reaction system. 1H and 13C NMR spectra of L-alanine formed in 2H2O have shown that an equimolar amount of [beta-2H1]- and [beta-2H2]alanines are produced. The deuterium isotope effect at the alpha position was observed; kH/kD = 2.4. These results indicated that the alpha hydrogen of selenocysteine was removed by a base at the active site, and was incorporated into the alpha position of alanine, a product, without exchange of a solvent deuterium. When the enzyme was incubated with L-selenocysteine in the absence of added pyridoxal 5'-phosphate, the activity decreased with prolonged incubation time. However, the activity was recovered by addition of 5'-phosphate. The spectrophotometric study showed that the inactivated enzyme was the apo form. The apoenzyme was activated by a combination of pyridoxamine 5'-phosphate and various alpha-keto acids such as alpha-ketoglutarate and pyruvate. Thus, the enzyme is inactivated through transamination between selenocysteine and the bound pyridoxal 5'-phosphate to produce pyridoxamine 5'-phosphate and a keto acid derived from selenocysteine. The pyridoxal enzyme, an active form, is regenerated by addition of alpha-keto acids. This regulatory mechanism is analogous to those of aspartate beta-decarboxylase [EC 4.1.1.12], arginine racemase [EC 5.1.1.9], and kynureninase [EC 3.7.1.3] [K. Soda and K. Tanizawa (1979) Adv. Enzymol. 49, 1].     

Biosynthetic origin of hydrogen atoms in the lipase inhibitor lipstatin

The lipase inhibitor lipstatin is biosynthesized in Streptomyces toxytricini via condensation of a C(14) precursor and a C(8) precursor, which are both obtained from fatty acid catabolism. To study the mechanism of this reaction in more detail, S. toxytricini was grown in medium containing a mixture of U-(13)C,U-(2)H-lipids and unlabeled sunflower oil or in a medium containing 70% D(2)O. Lipstatin was isolated and analyzed by (1)H,(2)H, and (13)C NMR spectroscopy. Hydrogen atoms at C-2, C-3, and C-4 of lipstatin were found to be derived from solvent protons. The formation of the lipstatin precursor 3-hydroxy-Delta(5,8)-tetradecadienoyl-CoA by beta oxidation of linoleic acid explains the incorporation of solvent hydrogen into the 4 position of lipstatin. The hydrogen in position 3 of lipstatin is most probably introduced from solvent by proton/deuterium exchange of a redox cofactor involved in the reduction of the keto group in the branched chain beta keto acid arising by a decarboxylative condensation. The incorporation of solvent hydrogen at position 2 can be explained by epimerization of a chiral intermediate at C-2 and C-3. Epimerization may involve a dehydration-rehydration mechanism.     

Deuterium Isotope Effect in γ-Aminobutyric Acid Transamination: Determination of Rate-Limiting Step

The rate of transamination of gamma-aminobutryic acid (GABA) catalyzed by hog brain gamma-aminobutyrate aminotransferase was substantially reduced when the hydrogen at the gamma-carbon position was replaced by deuterium. The deuterium isotope effect of this reaction has been substantiated by fluorometric, radiometric, and mass spectrometric procedures and assessed kinetically. The ratios of Vmax of the nonlabeled substrate/Vmax of the deuterated substrate obtained under different conditions ranged from 6 to 7. This indicates that the cleavage of the hydrogen from the gamma-carbon is the rate-determining step in GABA transamination. Similar isotope effects have also been shown to occur in the peripheral system in vivo.     

Stereochemistry of ornithine decarboxylase reaction

To determine the steric course of the reaction of bacterial ornithine decarboxylase [EC 4.1.1.17], we have carried out the decarboxylation of L-ornithine in 2H2O and that of DL-[2-2H]ornithine in H2O, and obtained putrescine bearing a single deuterium atom in the C-1 position. The stereochemistry of [1-2H]putrescine was established by conversion to 1-(2-pyrrolidinyl)-2-propanone with acetoacetate and the pro-S hydrogen-specific diamine oxidase from pea seedlings. Analysis of deuterium content by gas chromatography-mass spectrometry showed that the deuterium label was fully retained during the conversion of [1-2H]putrescine produced by the decarboxylation of L-ornithine in 2H2O to 1-(2-pyrrolidinyl)-2-propanone, in contrast with the considerable loss of label from [1-2H]putrescine which was produced by the decarboxylation of DL-[2-2H]ornithine in H2O. The extent of loss of the deuterium label was in good agreement with the estimated value based on the isotope effect in the diamine oxidase reaction. These results indicate that the introduced deuterium (or hydrogen) is in the pro-R position at C-1 of putrescine, and consequently the ornithine decarboxylase reaction proceeds with retention of configuration.     

Studies on the metabolism of unsaturated fatty acids. XII. Reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli

Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli (E. coli) was investigated. When trans-2,cis-4-decadienoyl-CoA was incubated with 4R- or 4S-[4-2H1]NADPH in the presence of purified 2,4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide. On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O, two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position. The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group. Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.     

Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin.

Using 4-methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter- and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent. With 4-trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron-transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate-limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C-H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron-transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.     

Peroxidase-catalyzed N-demethylation reactions: deuterium solvent isotope effects

The effect of D2O on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase was investigated in order to assess the roles of exchangeable hydrogens in the demethylation reaction. The initial rate of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide exhibited a pL optimum (where L denotes H or D) of 4.5 in both H2O and D2O. The solvent isotope effect on the initial rate of the chloroperoxidase-catalyzed demethylation reaction was independent of pL, suggesting that the solvent isotope effect is not due to a change in the pK of a rate-controlling ionization in D2O. The solvent isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction was 3.66 +/- 0.62. In contrast, the solvent isotope effect on the Vmax for the horseradish peroxidase catalyzed demethylation reaction was approximately 1.5 with either ethyl hydroperoxide or hydrogen peroxide as the oxidant, indicating that the exchange of hydrogens in the enzyme and hydroperoxide for deuterium in D2O has little effect on the rate of the demethylation reaction. The solvent isotope effect on the Vmax/KM for ethyl hydroperoxide in the chloroperoxidase-catalyzed demethylation reaction was 8.82 +/- 1.57, indicating that the rate of chloroperoxidase compound I formation is substantially decreased in D2O. This isotope effect is suggested to arise from deuterium exchange of the hydroperoxide hydrogen and of active-site residues involved in compound I formation. A solvent isotope effect of 2.96 +/- 0.57 was observed on the Vmax/KM for N,N-dimethylaniline in the chloroperoxidase-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma gonadotropin,prolactin levels and hypothalamic tyrosine hydroxylase activity in rats during estrous cycle,after overiectomy and after blockade of catecholamine biosynthesis

Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase activity were evaluated at 0900, 1200 and 1700 h during diestrus, proestrus and estrus, ovariectomized and after systemic administration of reserpine or α-methyl p-tyrosine, which interfere with catecholamine biosynthesis, in rats. Gonadotropin and prolactin levels showed peak values during the afternoon of proestrus, while hypothalamic tyrosine hydroxylase activity was markedly lowered at 1200 on proestrus. Gonadotropin levels were slightly lowered whereas prolactin concentrations and hypothalamic tyrosine hydroxylase activity were significantly increased by reserpine. Depletion of hypothalamic dopamine by reserpine apparently resulted in significant elevation of prolactin levels which inturn induce tyrosine hydroxylase. Gonadotropin levels and hypothalamic tyrosine hydroxylase activity were significantly suppressed after the administration of α-methyl p-tyrosine. Prolactin levels, however, were elevated significantly. These results indicate that catecholamines are involved in the control of gonadotropin and prolactin release during estrous cycle and inhibition of catecholamines biosynthesis by α-methyl p-tyrosine could result in suppression of gonadotropin levels, whereas removal of tonic inhibition of hypothalamic dopamine by α-methyl-p-tyrosine elevate prolactin levels.     

1H-n.m.r. studies of the isolated activation segment from pig procarboxypeptidase A.

The isolated activation segment (asA) from pig pancreatic procarboxypeptidase A was studied by 1H-n.m.r. spectroscopy over a wide range of solution conditions. Isolated asA shows many characteristics of compactly folded globular proteins, such as the observation of perturbed positions for resonances from methyl groups, alpha-carbon atoms, histidine residues and the tyrosine residue. The single tyrosine residue (Tyr-70) exhibits a very high pKa, and both histidine and tyrosine residues show slow chemical modification (deuteration and iodination). In contrast, asA shows rapid NH exchange. Analysis of the spectra by pH titration and nuclear Overhauser effects revealed several residue interactions. Quantitative analysis of deuterium and tritium exchange allowed the assignment of the histidine C-2-H resonances to their respective residues in the sequence. His-66, the closest to the sites of proteolytic attack in the proenzyme, is shown to be the most accessible to solvent in procarboxypeptidase A. It was also shown that asA is thermally very stable ['melting' temperature (Tm) 88 degrees C] and requires a high urea concentration for denaturation (6.25 M, at pH 7.5). Evidence is presented for some degree of conformational flexibility in the premelting range, a feature that could be ascribed to the preponderance of helical secondary structure and to the lack of disulphide bridges. The free solution structure of asA is probably unchanged when it binds to carboxypeptidase A.     

Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase

We have determined 15N isotope effects and solvent deuterium isotope effects for adenosine deaminase using both adenosine and the slow alternate substrate 7,8-dihydro-8-oxoadenosine. With adenosine, 15N isotope effects were 1.0040 in H2O and 1.0023 in D2O, and the solvent deuterium isotope effect was 0.77. With 7,8-dihydro-8-oxoadenosine, 15N isotope effects were 1.015 in H2O and 1.0131 in D2O, and the solvent deuterium isotope effect was 0.45. The inverse solvent deuterium isotope effect shows that the fractionation factor of a proton, which is originally less than 0.6, increases to near unity during formation of the tetrahedral intermediate from which ammonia is released. Proton inventories for 1/V and 1/(V/K) vs percent D2O are linear, indicating that a single proton has its fractionation factor altered during the reaction. We conclude that a sulfhydryl group on the enzyme donates its proton to oxygen or nitrogen during this step. pH profiles with 7,8-dihydro-8-oxoadenosine suggest that the pK of this sulfhydryl group is 8.45. The inhibition of adenosine deaminase by cadmium also shows a pK of approximately 9 from the pKi profile. Quantitative analysis of the isotope effects suggests an intrinsic 15N isotope effect for the release of ammonia from the tetrahedral intermediate of approximately 1.03 for both substrates; however, the partition ratio of this intermediate for release of ammonia as opposed to back-reaction is 14 times greater for adenosine (1.4) than for 7,8-dihydro-8-oxoadenosine (0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of acaterin: mechanism of the reaction catalyzed by dehydroacaterin reductase

Dehydroacaterin reductase is an enzyme which catalyzes the final step of acaterin biosynthesis, that is, the reduction of the C-4/C-5 double bond of dehydroacaterin. The mechanism of the reduction was investigated with a cell-free preparation obtained from the acaterin-producing microorganism, Pseudomonas sp. A 92. Incubation of dehydroacaterin in the presence of [4,4- 2H2]NADPH or D2O followed by 2H NMR analysis of the resulting acaterin revealed that the deuterium atom from NADPH was incorporated into the C-5 position of acaterin, while the deuterium atom from D2O was introduced into the C-4 position. It was further demonstrated that the pro-R hydrogen at C-4 of NADPH was stereospecifically utilized in this reduction.     

Energetics of proline racemase: double fractionation experiment, a test for concertedness and for transition-state dominance

To test whether a reaction involving the making and/or breaking of two bonds at two sites is concerted (and proceeds through a single transition state) or is stepwise (and involves a reaction intermediate in which only one bond has been made or broken), we have measured the isotopic fractionation at one site as a function of isotopic substitution at the other site. In the case of proline racemase, the discrimination against solvent deuterium in the product when the reaction is run in mixed H2O-D2O is measured for the reaction both of [2-1H]proline and of [2-2H]proline. The isotopic fractionation at the solvent site may in principle be smaller, the same, or larger, when the 2H-labeled substrate is used rather than the 1H substrate, and--depending upon the nature of the catalyzing groups--this information indicates whether the reaction is stepwise, or concerted, or whether an isotopically insensitive transition state is partially rate determining. Experimentally, we have found that the discrimination against solvent deuterium in the product L-proline is the same, whether D-[2-1H]proline or D-[2-2H]proline is the substrate. This result requires that the substrate and product "on-off" steps are faster than the racemization step and that the racemization reaction proceeds either in a concerted manner or in a stepwise fashion involving enzyme catalytic groups (e.g., thiols) having ground-state fractionation factors around 0.5.     

Solvent and solvent proton dependent steps in the galactose oxidase reaction

Solvent and solvent proton dependent steps involved in the mechanism of the enzyme galactose oxidase have been examined. The deuterium kinetic solvent isotope effect (KSIE) on the velocity of the galactose oxidase catalyzed oxidation of methyl beta-galactopyranoside by O2 was measured. Examination of the thermodynamic activation parameters for the reaction indicated that the isotope effect was attributable to a slightly less favorable delta H value, consistent with a KSIE on proton transfer. A detailed kinetic analysis was performed, examining the effect of D2O on the rate of reaction over the pH range 4.8-8.0. Both pL-rate profiles exhibited bell-shaped curves. Substitution of D2O as solvent shifted the pKes values for the enzymic central complex: pKes1 from 6.30 to 6.80 and pKes2 from 7.16 to 7.35. Analysis of the observed shifts in dissociation constants was performed with regard to potential hydrogenic sites. pKes1 can be attributed to a histidine imidazole, while pKes2 is tentatively assigned to a Cu2+-bound water molecule. A proton inventory was performed (KSIE = +1.55); the plot of kcat vs. mole fraction D2O was linear, indicating the existence of a single solvent-derived proton involved in a galactose oxidase rate-determining step (or steps). The pH dependence of CN- inhibition was also examined. The Ki-pH profile indicated that a group ionization, with pKa = 7.17, modulated CN- inhibition; Ki was at a minimum when this group was in the protonated state. The inhibition profile followed the alkaline limit of the pH-rate profile for the enzymic reaction, suggesting that the group displaced by CN- was also deprotonating above pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects

Mannitol dehydrogenases (MDH) are a family of Zn(2+)-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates. pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H(2)O and D(2)O at 25 degrees C and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion. The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K(mannitol)) and log k(cat) were 9.2 and 7.7 in H(2)O, respectively, and both were shifted by +0.4 pH units in D(2)O. Proton inventory plots for k(cat) and k(cat)/K(mannitol) were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level. They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H(2)O-D(2)O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K(mannitol) and k(cat), respectively. (D)(k(cat)/K(mannitol)) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of approximately 1. Measurement of (D)(k(cat)/K(fructose)) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction. A mechanism of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge. The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur. Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex. Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximately 85% rate-limiting for k(cat) and 2-fold smaller in D(2)O than in H(2)O.     

Chemical mechanism and rate-limiting steps in the reaction catalyzed by Streptococcus faecalis NADH peroxidase

The pH dependence of the kinetic parameters V, V/KNADH, and V/KH2O2 has been determined for the flavoenzyme NADH peroxidase. Both V/KNADH and V/KH2O2 decrease as groups exhibiting pK's of 9.2 and 9.9, respectively, are deprotonated. The V profile decreases by a factor of 5 as a group exhibiting a pK of 7.2 is deprotonated. Primary deuterium kinetic isotope effects on NADH oxidation are observed on V only, and the magnitude of DV is independent of H2O2 concentration at pH 7.5. DV/KNADH is pH independent and equal to 1.0 between pH 6 and pH 9.5, but DV is pH dependent, decreasing from a value of 7.2 at pH 5.5 to 1.9 at pH 9.5. The shape of the DV versus pH profile parallels that observed in the V profile and yields a similar pK of 6.6 for the group whose deprotonation decreases DV. Solvent kinetic isotope effects obtained with NADH or reduced nicotinamide hypoxanthine dinucleotide as the variable substrate are observed on V only, while equivalent solvent kinetic isotope effects on V and V/K are observed when H2O2 is used as the variable substrate. In all cases linear proton inventories are observed. Primary deuterium kinetic isotope effects on V for NADH oxidation decrease as the solvent isotopic composition is changed from H2O to D2O. These data are consistent with a change in the rate-limiting step from a step in the reductive half-reaction at low pH to a step in the oxidative half-reaction at high pH. Analysis of the multiple kinetic isotope effect data suggests that at high D2O concentrations the rate of a single proton transfer step in the oxidative half-reaction is slowed. These data are used to propose a chemical mechanism involving the pH-dependent protonation of a flavin hydroxide anion, following flavin peroxide bond cleavage.     

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.     

Deuterium oxide (D2O) enhances the photosensitivity of Stentor coeruleus.

Stentor coeruleus exhibits negative phototaxis and step-up photophobic response (avoiding reaction) to visible light (maximum at 610-620 nm in both responses). In the presence of deuterium oxide (D2O) the step-up photophobic response was markedly enhanced, whereas the phototactic orientation response was inhibited. The induction time for the step-up photophobic response was longer in D2O than in H2O, and the duration of ciliary reversal for the response was also longer in D2O than in H2O, indicating that certain steps of the sensory transduction chain are subject to solvent deuterium isotope effects. The enhancement of the step-up photophobic response in D2O was canceled by LaCl3, while the inhibition of the phototactic orientation response in D2O was partially removed by LaCl3, even though LaCl3 did not affect the phototactic orientation response. These results suggest that the sensory transduction mechanisms for the two photoresponses are different, although the photoreceptors (stentorin) are the same.     

Some aspects on L-dopa decarboxylase and p-tyrosine decarboxylase in the central nervous and peripheral tissues of the American cockroach Periplaneta americana

1. Aromatic amino acid decarboxylase activities toward L-DOPA (L-3,4-dihydroxyphenylalanine), 5-HTP (5-hydroxytryptophan) and p-tyrosine in different tissues of the sclerotized and newly ecdysed cockroach were analyzed. 2. The ratios of enzyme activity with regard to L-DOPA and p-tyrosine varied considerably in the tissues and between the two different growth stages. 3. A DOPA decarboxylase and a p-tyrosine decarboxylase were separated by gel filtration and ion exchange chromatography. 4. The optimal pH requirement for both enzymes was 7.5 with the exception of the one decarboxylating 5-HTP. 5. The molecular weights of the cockroach brain DOPA decarboxylase and tyrosine decarboxylase were estimated to be 120,000 and 100,000, respectively. 6. Unlike the mammalian aromatic amino acid decarboxylase, the cockroach DOPA decarboxylase cannot be activated by a small amount of benzene. 7. An increase of over 50-fold of DOPA decarboxylase activity and a 50% reduction of tyrosine decarboxylase activity in the epidermal tissue of the newly ecdysed animals was observed. 8. In the fully sclerotized cockroach, a reversible endogenous inhibitor(s) of DOPA decarboxylase in the integument was observed, suggesting that the DOPA decarboxylase is suppressed in the epidermal tissues when ecdysis does not occur.     

A new method for the measurement of protein turnover.

A new technique for the determination of rate constants of protein degradation is described. By using the method, half-lives of total soluble protein of Lemna minor during growth on full culture medium and distilled water were measured. The method involves incubating Lemna on a growth medium containing 3H2O. After a short exposure (20 min) to 3H-labelled culture medium, 3H was found in soluble amino acids, especially aspartate, glutamate, glutamine and alanine. After transfer to a 3H-free medium for 30 min, 80% of the 3H originally present in soluble amino acids was lost. These results suggest that 3H enters and leaves amino acids at the alpha-carbon atom, a conclusion supported by the observed labelling of glutamates. The exchange of H and 3H on the alpha-carbon atom is catalysed by transaminases and the speed of this exchange ensures that when the 3H2O is removed, the 3H in free amino acids is rapidly lost, thereby eliminating problems connected with metabolic pools and recycling. After an exposure of 20 min to 3H-labelled medium all protein amino acids, except for arginine, were found to be radioactive. The loss of radioactivity from protein amino acids was used to measure protein degradation.     

Studies with mechanism-based inactivators of lysine epsilon-transaminase from Achromobacter liquidum.

Analogues of lysine containing a 4,5-acetylenic linkage (lysyne) or a cis- or trans-4,5-olefinic linkage (lysenes) function as substrates for a homogeneous L-lysine epsilon-transaminase from Achromobacter liquidum but partition between transamination and time-dependent inactivation. The partition ratio is lowest for lysyne (40 per inactivation event) and higher for trans-lysene (160 per inactivation event), and the cis-lysene transaminates 1600 times per inactivation event. cis-Lysene yields alpha-picolinate as a detectable accumulating product, presumably from cyclization of initial 6-aldehyde to dihydropicolinate and spontaneous autoxidation. The trans isomer also yields some picolinate as an identifiable product. The product from the few lysyne turnovers is as yet unknown but has strong absorbance at 318 nm. The inactive enzyme species from all three lysine analogues slowly (overnight) regain full activity after gel filtration chromatography and dialysis, suggesting reversal of the initial adduct-forming reaction. Initial studies with partially purified pseudomonad lysine alpha-racemase show alpha-3H incorporation from 3H2O but no inactivation consistent with the expectation that these lysine analogues could act readily as mechanism-based inactivators for pyridoxal P enzymes which act at the epsilon- but not the alpha-carbon of lysine.