Cloning and heterologous expression of a broad specificity aminotransferase of Leishmania mexicana promastigotes1

We have previously reported that Leishmania mexicana promastigotes possess a broad substrate specificity aminotransferase (BSAT), able to transaminate aspartate, aromatic amino acids, methionine and leucine. We have confirmed now this unusual substrate specificity by cloning its gene and expressing in Escherichia coli the recombinant active protein. The amino acid sequence of BSAT shares over 40% identity with other eukaryotic and prokaryotic aspartate aminotransferases, thus showing that the enzyme belongs to the subfamily Ialpha of aminotransferases, and has only 6% identity with the tyrosine aminotransferase from Trypanosoma cruzi, which has a similar substrate specificity. The production of recombinant active enzyme in good yields opens up the possibility of obtaining its 3D-structure, in order to investigate the structural basis of the broad substrate specificity.     

Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode

The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group.     

Control of substrate specificity by active-site residues in nitrobenzene dioxygenase

Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the alpha subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics.     

Properties of the trihydroxytoluene oxygenase from Burkholderia cepacia R34: an extradiol dioxygenase from the 2,4-dinitrotoluene pathway

Burkholderia cepacia R34 mineralizes 2,4-dinitrotoluene via an oxidative pathway. The initial steps in the degradative pathway lead to formation of 2,4,5-trihydroxytoluene, which serves as the substrate for the ring cleavage dioxygenase. The trihydroxylated substrate differs from the usual substituted catechols found in pathways for aromatic compound degradation. To determine whether the characteristics of the trihydroxytoluene oxygenase reflect the unusual ring cleavage substrate of the 2,4-dinitrotoluene pathway, the gene encoding trihydroxytoluene oxygenase (dntD) was cloned and sequenced, and ring cleavage activity determined from recombinant bacteria carrying the cloned gene. The findings were compared to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT and to other previously described ring cleavage dioxygenases. The comparison revealed that only 60% identity was shared by the two trihydroxytoluene oxygenases, but the amino acid residues involved in cofactor binding, catalysis, and protein folding were conserved in the DntD sequence. The enzyme catalyzed meta-fission of trihydroxytoluene as well as the substrate analogues 1,2,4-benzenetriol, catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and 2,3-dihydroxybiphenyl. However, results from enzyme assays indicated a strong preference for trihydroxytoluene, implying that it was the native substrate for the enzyme. The apparent enzyme specificity, its similarity to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT, and the distant genetic relationship to other ring cleavage enzymes suggest that dntD evolved expressly to carry out trihydroxytoluene transformation.     

Intestinal plurispecific aromatic aminotransferase.

Aromatic: 2-oxoglutarate aminotransferase has been purified about 680-fold from the extracts of rat small intestine. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis. On the basis of substrate specificity, substrate inhibition, polyacrylamide gel electrophoresis and other some properties of this enzyme, it has been suggested that tyrosine: 2-oxoglutarate aminotransferase is identical with phenylalanine and kynurenine: 2-oxoglutarate amino-transferases, and also with aspartate: 2-oxoglutarate aminotransferase.     

Destabilization of tyrosine aminotransferase by amino acids

Summary Several L-amino acids (tyrosine, glutamate, methionine, tryptophan, and phenylalanine) and penicillamine destabilized purified tyrosine aminotransferase by removing enzyme-bound pyridoxal 5-phosphate. The destabilization was measured as a progressive loss of enzyme activity in samples taken at intervals from a primary mixture that was incubated at 37°C. Each destabilizing amino acid either served as a substrate for this enzyme or was a product of transamination. In contrast, L-cysteine destabilized the enzyme only if liver homogenate was added, which generated polysulfide by desulfuration. Cysteine complexed free pyridoxal-5-phosphate but did not remove it from the enzyme. Other amino acids did not destabilize tyrosine aminotransferase at the concentrations tested.Abbreviations TyrAT tyrosine aminotransferase (E.C. 2.6.1.5) - PLP pyridoxal-5-phosphate     

Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar K_m for the substrate based on product formation rates and whole-cell kinetics.     

Acceleration of the degradation of tyrosine aminotransferase in rat hepatome (HTC) cells by inhibitors of cyclic nucleotide phosphodiesterase.

The following inhibitors of cyclic nucleotide phosphodiesterase reduce the specific activity of tyrosine aminotransferase when added to cultures of rat hepatoma (HTC) cells: theophylline, 1-methyl-3-isobutylxanthine, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, and papaverine. Immunochemical measurements show that each inhibitor reduces the rate of tyrosine aminotransferase synthesis and increases the rate of degradation of the enzyme. The effect on synthesis also occurs with general proteins while that on degradation appears specific for tyrosine aminotransferase.     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

CEREBRAL AROMATIC AMINOTRANSFERASE

—Aromatic: 2-oxoglutarate aminotransferase has been purified about 950-fold from rat brain mitochondria. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis and had a molecular weight of approx 63,000. On the basis of substrate specificity, substrate inhibition, purification ratio, yield, polyacrylamide gel electrophoresis and some other properties of the enzyme it has been suggested that brain mitochondrial tyrosine:2-oxoglutarate aminotransferase (l -tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) is identical with brain mitochondrial phenylalanine and kynurenine: 2-oxoglutarate aminotransferases (l -kynurenine: 2-oxoglutarate aminotransferase, EC 2.6.1.7), and also with aspartate: 2-oxoglutarate aminotransferase (l -aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1).     

The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin and non-heme iron-dependent enzyme that hydroxylates L-Phe to l-Tyr using molecular oxygen as additional substrate. A dysfunction of this enzyme leads to phenylketonuria (PKU). The conformation and distances to the catalytic iron of both L-Phe and the cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) simultaneously bound to recombinant human PAH have been estimated by (1)H NMR. The resulting bound conformers of both ligands have been fitted into the crystal structure of the catalytic domain by molecular docking. In the docked structure L-Phe binds to the enzyme through interactions with Arg270, Ser349 and Trp326. The mode of coordination of Glu330 to the iron moiety seems to determine the amino acid substrate specificity in PAH and in the homologous enzyme tyrosine hydroxylase. The pterin ring of BH2 pi-stacks with Phe254, and the N3 and the amine group at C2 hydrogen bond with the carboxylic group of Glu286. The ring also establishes specific contacts with His264 and Leu249. The distance between the O4 atom of BH2 and the iron (2.6(+/-0.3) A) is compatible with coordination, a finding that is important for the understanding of the mechanism of the enzyme. The hydroxyl groups in the side-chain at C6 hydrogen bond with the carbonyl group of Ala322 and the hydroxyl group of Ser251, an interaction that seems to have implications for the regulation of the enzyme by substrate and cofactor. Some frequent mutations causing PKU are located at residues involved in substrate and cofactor binding. The sites for hydroxylation, C4 in L-Phe and C4a in the pterin are located at a distance of 4.2 and 4.3 A from the iron moiety, respectively, and at 6.3 A from each other. These distances are adequate for the intercalation of iron-coordinated molecular oxygen, in agreement with a mechanistic role of the iron moiety both in the binding and activation of dioxygen and in the hydroxylation reaction.     

Effects of C5 protein on Escherichia coli RNase P catalysis with a precursor tRNA(Phe) bearing a single mismatch in the acceptor stem

Escherichia coli RNase P, an RNA-processing enzyme that cleaves precursor tRNAs to generate the mature 5'-end, is composed of a catalytic component (M1 RNA) and a protein cofactor (C5 protein). In this study, effects of C5 protein on the RNase P catalysis with a precursor E. coli tRNA(Phe) having a single mismatch in the acceptor stem were examined. This mutant precursor unexpectedly generated upstream cleavage products at the -8 position as well as normal cleavage products at the +1 position. The cleavage at the -8 position was essentially effective only in the presence of C5 protein. Possible secondary structures for cleavage at the -8 position deviate significantly from the structures of the known RNase P substrates, implying that C5 protein can allow the enzyme to broaden the substrate specificity more than previously appreciated.     

Crystal structure of rabbit phosphoglucose isomerase complexed with its substrate D-fructose 6-phosphate.

Phosphoglucose isomerase (PGI, EC 5.3.1.9) catalyzes the interconversion of D-glucose 6-phosphate (G6P) and D-fructose 6-phosphate (F6P) and plays important roles in glycolysis and gluconeogenesis. Biochemical characterization of the enzyme has led to a proposed multistep catalytic mechanism. First, the enzyme catalyzes ring opening to yield the open chain form of the substrate. Then isomerization proceeds via proton transfer between C2 and C1 of a cis-enediol(ate) intermediate to yield the open chain form of the product. Catalysis proceeds in both the G6P to F6P and F6P to G6P directions, so both G6P and F6P are substrates. X-ray crystal structure analysis of rabbit and bacterial PGI has previously identified the location of the enzyme active site, and a recent crystal structure of rabbit PGI identified Glu357 as a candidate functional group for transferring the proton. However, it was not clear which active site amino acid residues catalyze the ring opening step. In this paper, we report the X-ray crystal structure of rabbit PGI complexed with the cyclic form of its substrate, D-fructose 6-phosphate, at 2.1 A resolution. The location of the substrate relative to the side chains of His388 suggest that His388 promotes ring opening by protonating the ring oxygen. Glu216 helps to position His388, and a water molecule that is held in position by Lys518 and Thr214 accepts a proton from the hydroxyl group at C2. Comparison to a structure of rabbit PGI with 5PAA bound indicates that ring opening is followed by loss of the protonated water molecule and conformational changes in the substrate and the protein so that a helix containing amino acids 513-520 moves in toward the substrate to form additional hydrogen bonds with the substrate.     

Determinants of substrate specificity in omega-aminotransferases

Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the alpha-carboxylate of alpha-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the delta-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.     

Initial reactions in the anaerobic oxidation of toluene and m-xylene by denitrifying bacteria.

Pseudomonas sp. strain T and Pseudomonas sp. strain K172 grow with toluene under denitrifying conditions. We demonstrated that anaerobic degradation of toluene was initiated by direct oxidation of the methyl group. Benzaldehyde and benzoate accumulated sequentially after toluene was added when cell suspensions were incubated at 5 degrees C. Strain T also grows anaerobically with m-xylene, and we demonstrated that degradation was initiated by oxidation of one methyl group. In cell suspensions incubated at 5 degrees C 3-methylbenzaldehyde and 3-methylbenzoate accumulated after m-xylene was added. Toluene- or m-xylene-grown strain T cells were induced to the same extent for oxidation of both hydrocarbons. In addition, the methyl group-oxidizing enzyme system of strain T also catalyzed the oxidation of each isomer of the chloro- and fluorotoluenes to the corresponding halogenated benzoate derivatives. In contrast, strain K172 only oxidized 4-fluorotoluene to 4-fluorobenzoate, probably because of the narrow substrate specificity of the methyl group-oxidizing enzymatic system. During anaerobic growth with toluene strains T and K172 produced two transformation products, benzylsuccinate and benzylfumarate. About 0.5% of the toluene carbon was converted to these products.     

Gabaculine resistance of Synechococcus glutamate 1-semialdehyde aminotransferase.

Glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes the transfer of the C2 amino group of glutamate 1-semialdehyde (GSA) to the C1 position. Nucleic acid sequences encoding this enzyme from wild type and a gabaculine (GAB) resistant strain of Synechococcus have been cloned and overexpressed in Escherichia coli. Tolerance to GAB of the mutant GSA-AT resulted from a point mutation, Met-248-Ile, in the middle of the polypeptide chain accompanied by a deletion of three amino acids close to the NH2 terminus but can also be effected by the point mutation alone. Purified enzymes from these two strains contain vitamin B6 and use a typical ping-pong Bi-Bi mechanism, in which 4,5-diaminovalerate (DAVA) is a likely intermediate. The catalytic efficiency (Kcat/Km) of wild-type GSA-AT for GSA is about 3 times larger than that of the mutant enzyme. Comparison of substrate specificities (kmax/Km) for GSA and various analogues reveals that wild-type GSA-AT has values that are about 2-20 times larger than those of the mutant enzyme, except in the case of GAB for which the specificity is 2-3 orders of magnitude larger. These differences are attributed to impaired prototropic rearrangement and transaldimination by mutant GSA-AT. They lead to accumulation of quinonoid and other intermediates upon addition of various substrates such as ALA and DOVA, as well as to instability of their aldimines (418 nm) upon Sephadex gel filtration.     

Construction of a 3-chlorobiphenyl-utilizing recombinant from an intergeneric mating.

Recombinant Pseudomonas sp. strain CB15, which grows on 3-chlorobiphenyl (3CB), was constructed from Pseudomonas sp. strain HF1, which grows on 3-chlorobenzoate, and from Acinetobacter sp. strain P6, which grows on biphenyl, by using a continuous amalgamated culture apparatus. DNA from strains CB15 and HF1 hybridized very strongly to each other, while hybridization between both parental strains, HF1 and P6, was negligible. However, DNA from the recombinant CB15 hybridized moderately to strongly with three specific fragments of parental strain P6. Strains HF1 and P6 did not grow on 3CB, but recombinant strain CB15 mineralized this compound and released inorganic chloride. When growing on 3CB, strain CB15 accumulated brown products, one of which was identified as 3-chloro-5-(2'-hydroxy-3'-chlorophenyl)-1,2-benzoquinone by mass spectrometry. Emulsification and mechanical fragmentation greatly increased the rate of 3CB mineralization by strain CB15. At least three methods of inhibition from catecholic intermediates may account for slow growth on 3CB. The meta fission of 2,3-dihydroxybiphenyl (the nonchlorinated analog of the metabolic intermediate 3-chloro-2',3'-dihydroxybiphenyl) was affected by substrate inhibition (Vmax = 359 nmol.min-1.mg-1, Km = 114 microM, Kss [the inhibition constant] = 951 microM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmol.min-1.mg-1, Km = 1 microM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of a 3-chlorobiphenyl-utilizing recombinant from an intergeneric mating.

Recombinant Pseudomonas sp. strain CB15, which grows on 3-chlorobiphenyl (3CB), was constructed from Pseudomonas sp. strain HF1, which grows on 3-chlorobenzoate, and from Acinetobacter sp. strain P6, which grows on biphenyl, by using a continuous amalgamated culture apparatus. DNA from strains CB15 and HF1 hybridized very strongly to each other, while hybridization between both parental strains, HF1 and P6, was negligible. However, DNA from the recombinant CB15 hybridized moderately to strongly with three specific fragments of parental strain P6. Strains HF1 and P6 did not grow on 3CB, but recombinant strain CB15 mineralized this compound and released inorganic chloride. When growing on 3CB, strain CB15 accumulated brown products, one of which was identified as 3-chloro-5-(2'-hydroxy-3'-chlorophenyl)-1,2-benzoquinone by mass spectrometry. Emulsification and mechanical fragmentation greatly increased the rate of 3CB mineralization by strain CB15. At least three methods of inhibition from catecholic intermediates may account for slow growth on 3CB. The meta fission of 2,3-dihydroxybiphenyl (the nonchlorinated analog of the metabolic intermediate 3-chloro-2',3'-dihydroxybiphenyl) was affected by substrate inhibition (Vmax = 359 nmol.min-1.mg-1, Km = 114 microM, Kss [the inhibition constant] = 951 microM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmol.min-1.mg-1, Km = 1 microM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine aminotransferase activity of frog (Rana esculenta) liver

1. The presence of tyrosine aminotransferase is reported both in particulate and soluble fractions of frog liver. 2. The activity of the soluble enzyme of frog liver was investigated with regard to its dose and time dependence, its substrate specificity and concentration dependence, its thermal sensitivity as well as pH and temperature dependence. 3. It appears that the properties of the soluble tyrosine aminotransferase of frog liver are in close agreement with those reported for the mammalian liver enzyme.     

Possible involvement of cGMP in the control of tyrosine aminotransferase degradation in rat hepatocytes

Addition of theophylline to primary cultures of rat hepatocytes in which tyrosine aminotransferase had been preinduced with dexamethasone caused a further increase in specific activity of the enzyme. This increase was due in part to a reduction in the rate of tyrosine aminotransferase degradation that began about 2 hr after theophylline was added. The level of cGMP also increased with a similar time lag following the addition of theophylline. The concentration of theophylline which produced the above effects (1 mM) did not alter the rate of general protein degradation in hepatocytes. Addition of 8-bromo-cGMP (0.5 mM) resulted in an immediate reduction in the rate of tyrosine aminotransferase degradation and in an increase in the activity of the enzyme. Treating hepatocytes with MnCl2 (0.9 mM) caused an elevation of cGMP and a concomitant slowing of tyrosine aminotransferase degradation without changing the level of cAMP significantly. These results suggest an inverse relationship between the level of cGMP and the rate of tyrosine aminotransferase degradation in hepatocytes.