Partial purification and substrate specificity of flavin-containing monooxygenase from rat brain microsomes

Flavin-containing monooxygenase (FMO) was partially purified from rat brain microsomes through two successive chromatographies on columns of DEAE Sepharose and 2',5'-ADP Sepharose. The specific activity, benzydamine N-oxidation of partially purified brain FMO, was 122-fold higher than that of microsomes. A single band of 60 kDa was recognized by Western blotting analysis with anti-rat liver FMO. The Km value of brain FMO for thiourea was 4-fold lower, but that for cysteamine was 10-fold higher than that of liver FMO. The enzymatic activity for n-octylamine was detected in neither brain nor liver FMO. Kinetic analysis for neurotoxins also revealed that Km values of brain FMO for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and N-methyl TIQ (NMTIQ) were lower than those of liver FMO. These results indicate that rat brain FMO catalyzes several substrates of liver FMO involving neurotoxins, but it seems likely that the kinetic properties of brain FMO are somewhat different from those of liver FMO.     

An assay of flavin-containing monooxygenase activity with benzydamine N-oxidation

An assay system of flavin-containing monooxygenase was developed by fluorometric determination of benzydamine (BZY) N-oxidation with HPLC. The apparent Km value for the formation of BZY N-oxide from BZY by rat liver microsomes was similar to that by purified FMO. The Km and Vmax values for the formation of N-desmethylbenzydamine (Nor-BZY) by rat liver microsomes were about 50 times greater and 2000 times less, respectively, than those of BZY N-oxide. Nor-BZY was not formed upon incubation with purified enzyme. BZY N-oxidation activity was completely inhibited both in the absence of NADPH and by heat inactivation. The reaction was inhibited in the presence of 0.5 mM thiourea, but 2 mM SKF-525A did not affect BZY N-oxidation. Moreover, rabbit antibody raised against the rat enzyme inhibited BZY N-oxidation. These results are in accord with a simple, rapid, and sensitive assay for the enzyme.     

Absence of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by flavin-containing monooxygenase (FMO)

The N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco and tobacco smoke. Carbonyl reduction, alpha-carbon hydroxylation (activation) and N-oxidation of the pyridyl ring (detoxification) are the three main pathways of metabolism of NNK. In this study, metabolism of NNK was studied with lung and liver microsomes from F344 rats, Syrian golden hamsters and pigs and cloned flavin-containing monooxygenases (FMOs) from human and rabbit liver. Thermal inactivation at 45 degrees C for 2 min reduced FMO S-oxygenating activity but did not affect N-oxidation of NNK, leading to the conclusion that FMOs are not implicated in the detoxification of NNK. Detoxification of NNK was not increased by n-octylamine or by incubation at pH 8.4, supporting the conclusion that FMOs are not involved in the metabolism of NNK. SKF-525A (1 mM) significantly reduced N-oxidation and alpha-carbon hydroxylation, suggesting that these two pathways were catalyzed by cytochromes P450. Metabolism of NNK was lower with lung microsomes than with liver microsomes. Inhibition of metabolism of NNK by SKF-525A was also observed with rat lung microsomes, leading to the conclusion that cytochromes P450 are involved in pulmonary metabolism of NNK. Cloned FMOs did not metabolize NNK. In conclusion, cytochromes P450 rather than FMOs are involved in N-oxidation of NNK. The high capacity of hamster liver microsomes to activate NNK does not correlate with the resistance of this tissue to NNK-induced hepatocarcinogenesis.     

Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3

In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.     

Identification of amino acid residues associated with modulation of flavin-containing monooxygenase (FMO) activity by imipramine: structure/function studies with FMO1 from pig and rabbit

The activity of the flavin-containing monooxygenase (FMO) can be modulated by a number of nitrogen-containing compounds in a manner that is both isoform and modulator-dependent. We now show that the direction (activation or inhibition) and extent of modulation can also be dependent on substrate concentration. Imipramine activates methimazole metabolism catalyzed by rabbit FMO1 or FMO2 at methimazole concentrations greater than 50 or 100 microM, respectively, and inhibits at lower methimazole concentrations. The extent of the activation increases as the substrate concentration increases, and the extent of inhibition increases as the substrate concentration decreases. With either inhibition or activation, the magnitude of the effect shows a similar, direct dependency on imipramine concentration. In contrast, imipramine inhibits the metabolism of methimazole catalyzed by pig FMO1 at all substrate concentrations. The structural basis for this unique ortholog difference between the responses of rabbit and pig FMO1 to imipramine was studied by random chimeragenesis and site-directed mutagenesis. Results with chimeras indicated that modulation of FMO1 activity by imipramine is controlled to a great extent by two areas of the FMO primary structure (residues 381-432 and 433-465). Four amino acids in these regions (positions 381, 400, 420 and 433) and one additional residue (position 186) were identified by site-directed mutagenesis as primary determinants of the imipramine response. When the residues at these positions in rabbit FMO1 are exchanged for the corresponding residues of pig FMO1, a mutant with the functional properties of pig FMO1 is produced. Our results suggest that the response of FMO1 to imipramine involves a distribution between two sites that is regulated by structural features that do not alter the overall binding. The inhibition observed, although it appears to be competitive, likely does not involve competition for a binding site since alteration of imipramine metabolism has no effect on the parameters of methimazole metabolism.     

A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose. Implications for a proposed mechanism of H+/lactose symport

In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with Km values of 0.42 and 1.30 mM and Vmax values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal Km of 0.36 mM and a moderate Vmax of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal Km of 0.27 mM but a very defective Vmax of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the Vmax values were lower for the Ala-319 mutations. When comparing the Vmax values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in Vmax caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar-dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.     

Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.     

Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit

The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.     

Biochemical characterization of a human band 4.1-related protein-tyrosine phosphatase, PTPH1

PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeleton-associated proteins. Here, we report the purification and biochemical characterization of this enzyme from baculovirus-infected insect cells. The purified protein exhibited an apparent M(r) of 120,000 on SDS gels. The native enzyme dephosphorylated both myelin basic protein (MBP) and reduced, carboxamidomethylated, and maleylated lysozyme (RCML) but was over 5-fold more active on MBP. The Km values for the two substrates were similar (1.45 microM for MBP and 1.6 microM for RCML). Phosphorylation of PTPH1 by protein kinase C in vitro resulted in a decrease in Km but had no effect on Vmax. Removal of the NH2-terminal band 4.1 homology domain of PTPH1 by limited trypsin cleavage stimulated dephosphorylation of RCML but inhibited its activity toward MBP. The dephosphorylation of RCML by full-length PTPH1 was enhanced up to 6-fold by unphosphorylated MBP and increasing ionic strength up to 0.2 M NaCl, whereas trypsinized preparations of PTPH1 containing the isolated catalytic domain were unaffected. These results suggest that in addition to a potential role in controlling subcellular localization, the NH2-terminal band 4.1 homology domain of PTPH1 may exert a direct effect on catalytic function.     

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.     

S-oxidation of (+)-cis-3,5-dimethyl-2-(3-pyridyl)-thiazolidin-4-one hydrochloride by rat hepatic flavin-containing monooxygenase 1 expressed in yeast

1. Rat hepatic flavin-containing monooxygenase 1 (FMO1) expressed in yeast catalyzed the S-oxidation of (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) in vitro. 2. S-oxidation was inhibited by 1-(1-naphthyl)-2-thiourea and thiobenzamide, known inhibitors of FMO, but was not enhanced by n-octylamine, a known enhancer of FMO. 3. The rate of S-oxide formation from SM-12502 was about four-fold lower than that from (+/-)-trans-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-9979) and enantioselectivity and diastereoselectivity of the S-oxidation reaction were observed. 4. The ability of the recombinant yeast to produce the S-oxide from SM-12502 was maintained for long periods and exemplifies the recombinant yeast as a bioreactor to produce a large amount of the S-oxide.     

Major pathway of imipramine metabolism is catalyzed by cytochromes P-450 1A2 and P-450 3A4 in human liver

The metabolism of imipramine by human liver microsomes was examined using a combination of five strategies. Human hepatic microsomes produced N-desmethylimipramine (84%), 2-hydroxyimipramine (10%), and 10-hydroxyimipramine (6%). Preincubation of human hepatocytes in culture with beta-naphthoflavone and macrolides exclusively induced the formation of desmethylimpramine (552%, p < 0.05, and 234%, p < 0.003, respectively). Correlations were obtained between rates of imipramine demethylation and cytochrome P-450 (P-450) 1A2 (r = 0.88, p < 0.001) and P-450 3A (r = 0.80, p < 0.02) concentrations in human liver microsomal preparations from 13 different subjects. Anti-P-450 1A2 and anti-P-450 3A antibodies selectively inhibited N-demethylation (80% and 60%, respectively). N-Demethylation was completely inhibited when anti-1A2 and anti-3A were added simultaneously. Kinetic studies with human microsomes confirm the contribution of two different enzymes in the N-demethylation. The Km of 1A2 was similar to the high affinity Km in human liver microsomes, whereas the Km of 3A was similar to the low affinity Km in human liver microsomes. P-450 1A2 was apparently more efficient than 3A4 (lower Km and higher Vmax) but was expressed in much lower concentration. Human P-450s 1A2 and 3A4 expressed in yeast efficiently produced desmethylimipramine. These results suggest that P-450 1A2 and P-450 3A4 are the major enzymes involved in imipramine N-demethylation in human hepatic microsomes. Similar experiments were conducted using P-450 2D6, and they confirmed that P-450 2D6 catalyzes imipramine 2-hydroxylation. Interindividual variations in 3A4 hepatic content may explain the large variations in imipramine blood levels observed after uniform dosages and thus may explain the variations in clinical efficacy. Caution might be advised in the clinical use of tricyclic antidepressants when drugs are also administered that induce or inhibit P-450s 3A4 and 1A2.     

Enzymatic reduction of chromium(VI) by human hepatic microsomes

The reduction of chromium(VI) by human hepatic microsomes was investigated. The reduction rates were proportional to the amount of microsomes added and reduction was mediated by an NADPH-dependent enzymatic system which exhibited a Km for chromate of 1.04 +/- 0.18 microM and a Vmax of 5.03 +/- 0.49 nmol/min/mg protein. Relative to incubation under 0% O2, 21% O2 inhibited microsomal Cr(VI) reduction in three individuals by 53, 36 and 37%. Cr(VI) reduction was not inhibited by metyrapone, carbon monoxide, aminopyrine, piperonyl butoxide or chloroform, suggesting that cytochrome P450s did not play a major role. Thallium trichloride (0.13 and 0.26 mM), a known flavoprotein inhibitor, caused a complete inhibition of both Cr(VI) reduction and NADPH:cytochrome P450 (c) reductase activity. A partial inhibition of Cr(VI) reduction was seen in the presence of n-octylamine, which may suggest a possible role for flavin-containing monooxygenase (FMO). Overall, human microsomal Cr(VI) reduction is very different from the P450-mediated microsomal reduction observed in rodents. Specifically, the human system is much less oxygen-sensitive, has a much greater affinity for chromate and is apparently mediated by flavoproteins.     

Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant. There were significant increases in the Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax value for the F132H/F306H mutant was increased to 3100 s-1. These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.     

Effects of L-histidine and its structural analogues on human N-myristoyltransferase activity and importance of EEVEH amino acid sequence for enzyme activity

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Human NMT (hNMT) activity was found to be activated by L-histidine in a concentration-dependent manner. In contrast, two structural analogues of L-histidine, L-histidinol and histamine, inhibited hNMT activity in a noncompetitive manner with half-maximal inhibitions of 18 and 1.5 mM, respectively. The inhibition of hNMT activity by L-histidinol was reversed by a 2-fold molar excess of L-histidine, suggesting that L-histidine and L-histidinol were competing for a common site on NMT. Kinetic data indicated that whereas L-histidine enhanced the Vmax, both L-histidinol and histamine decreased the Vmax; none of these compounds altered the Km. Our studies suggest that L-histidine and its analogues may be interacting with His-293, involved in myristoyl-CoA transfer, rather than His-218, and implicated in the transfer of myristoyl-CoA to the peptide substrates. Site-directed mutagenesis of His-293, Val-291, and Glu-290 resulted in proteins with no measurable NMT activity. The most conserved region in the catalytic domain EEVEH (289-293) is critical for the myristoyl-CoA transfer in the NMT-catalyzed reactions. This region will be useful for the design of regulators of NMT function.     

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human cytochrome P4501A1, P4501A2 and P4501B1

While the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by N-hydroxylation has been well documented, the relative roles of the human cytochrome P450 (CYP) enzymes that catalyze this reaction have not been established. Previous studies indicated that the mutagenic activation product, 2-hydroxyamino-PhIP (N2-OH-PhIP), is produced primarily by CYP1A2, and to a lesser extent by CYP1A1. We recently reported that human CYP1B1 also produces N2-OH-PhIP (Carcinogenesis, 18, 1793-1798, 1997). In the present study, we examined PhIP metabolism by microsomes containing recombinant human CYP1A1, 1A2 or 1B1 expressed in Sf9 insect cells and compared the kinetic values for PhIP metabolite formation. PhIP metabolites were analyzed by high pressure liquid chromatography with fluorescence and absorbance detection. Vmax values for N2-OH-PhIP formation were 90, 16 and 0.2 nmol/min/nmol P450, and the apparent Km values were 79, 5.1 and 4.5 microM for human CYP1A2, 1A1 and 1B1, respectively. The non-mutagenic metabolite, 4'-hydroxy-PhIP, was also formed by all three CYP enzymes with Vmax values of 1.5, 7.8 and 0.3 nmol/ min/nmol P450 and apparent Km values of 43, 8.2 and 2.2 microM for human CYP1A2, 1A1 and 1B1, respectively. Although the Vmax for N2-OH-PhIP production was highest for CYP1A2, the catalytic efficiency (Vmax/Km) of CYP1A1 was greater than that of CYP1A2. These results suggest that, for humans, extrahepatic CYP1A1 may be more important than previously thought for the metabolic activation of the dietary carcinogen PhIP.     

The role of the active site cysteine in catalysis by type 1 iodothyronine deiodinase

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mM concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in K(i)propylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mM reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.     

Inhibitory effects of novel nucleoside and nucleotide analogues on Epstein-Barr virus replication

Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently complex. Algebraic expressions for the primary kinetic parameters (Km and Vmax) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport system such as the Na+/alanine cotransporter in LLC-PK1 cells (1:1 Na+ to substrate coupling, and an ordered binding sequence), the algebraic expressions for Km for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here that the expression of Km of the first-bound substrate (Na+) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes the rate limiting step. Under these conditions, the expression for the KNam includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na+, and (ii) conformational 'translocation' of the substrate-free protein. The influence of imposed changes in membrane potential on the apparent KNam for the LLC-PK1 alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential dependence for KNam at 5 micron alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport system.     

Isolation and certain properties of mutant alkaline phosphatase of Escherichia coli

Natural and mutant alkaline phosphatases with amino acid substitutions in the processing site and N-terminal domain of the mature polypeptide chain Val for Ala(-1), Gln for Glu (+4) and simultaneously Gln for Glu (+4) and Ala for Arg (+1) have been isolated from the periplasm and cultural fluid of E. coli. It has been found that these substitutions have little effect on the dependence of the enzyme activity on pH, ionic strength and temperature but influence its isoenzymic spectrum and decrease (almost twofold) the maximal rate of the enzyme-catalyzed reaction. Extracellular enzymes display, in contrast with periplasmic ones, other catalytic properties (Vmax) and binding activity (Km). After translocation through the outer membrane all the enzymes display decreased Vmax and increased Km. These changes are especially well-pronounced in case of the mutant protein PhoA46 which contains an uncleaved signal peptide due to the impossibility of processing resulting from the substitution of Val for Ala(-1). The Vmax for this protein is decreased 20 times, while the Km is increased 4-fold. The protein also shows a higher (in comparison with other proteins) sensitivity towards proteolytic enzymes and is less resistant upon storage. The experimental data suggest that the changes in the N-end of alkaline phosphatase located at a long distance from its active center influence the enzyme function.     

Negative regulation of Raf-1 by phosphorylation of serine 621

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.