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S Ghisla

Publications and source records attributed to S Ghisla.

At least 55 records · Page 3Linked to original sources
PubMed1995

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes.

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].

Base Sequence↗
PubMed1995

Kinetic and mechanistic studies on the reactions of 2-aminobenzoyl-CoA monooxygenase/reductase.

The kinetic mechanism of the flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase with its natural substrates 2-aminobenzoyl-CoA, NADH and O2 has been investigated using the stopped-flow technique. Initial rate measurements indicate the formation of a ternary complex between oxidized enzyme and the two substrates 2-aminobenzoyl-CoA and NADH, a turnover number of approximately 40 min-1 was found at pH 7.4 and 4 degrees C. 2-Aminobenzoyl-CoA binds to oxidized enzyme to form a complex which is in a approximately 1:1 equilibrium with a second, spectrophotometrically distinguishable one. Binding of 2-amino benzoyl-CoA to reduced enzyme is, in contrast, a simple second-order process. Reduction of oxidized enzyme, both uncomplexed and in complex with 2-aminobenzoyl-CoA, by NADH is strongly biphasic. The first fast phase yields enzyme in which 50% of the total FAD is reduced to the FADH2 state. This rate is not affected by the presence of 2-aminobenzoyl-CoA. In contrast, 2-aminobenzoyl-CoA enhances approximately 100-fold the second phase, the reduction of the residual 50% FAD. This second phase of reduction (kobs = 2.0 s-1) is partially rate-limiting in catalysis. The oxygen reaction of uncomplexed, reduced enzyme is also biphasic and no oxygenated intermediate was detected. Reoxidation of substrate-complexed, reduced enzyme involves three spectroscopically distinguishable species. The first observable intermediate is highly fluorescent suggesting that it consists largely of flavin-4a-hydroxide. Thus, insertion of oxygen into 2-aminobenzoyl-CoA is essentially complete at this point and has a kobs > or = 80 s-1. The subsequent phase is accompanied by formation of the main product, 2-amino-5-oxocyclohex-1-enecarboxyl CoA. This step consists in a hydrogenation of the primary, oxygenated and non-aromatic CoA intermediate; it has a rate approximately 1.3 s-1, which is thus the second rate-limiting step in catalysis. As a side reaction of the oxidized enzyme and at low NADH concentrations the initially formed product disappears at a very slow rate (kobs approximately 0.05 s-1). This third 'post-catalytic' process is not relevant for catalysis. The primary product 2-amino-5-oxocyclohex-1-enecarboxyl-CoA is dehydrogenated by the oxidized enzyme to yield the aromatic 2-amino-5-hydroxybenzoyl-CoA as secondary product. The reduced enzyme formed in this process is reoxidized by O2 to form H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics↗
PubMed1995

2-Aminobenzoyl-CoA monooxygenase/reductase. Evidence for two distinct loci catalyzing substrate monooxygenation and hydrogenation.

2-Aminobenzoyl-CoA monooxygenase/reductase catalyzes both monooxygenation and hydrogenation of anthraniloyl-CoA. Its reactivity with 11 substrate analogs has been investigated. Only 2-aminobenzoyl-CoA (anthraniloyl-CoA) in its normal and deuterated (5-2H) form is a full substrate, and only traces of 2-hydroxybenzoyl-CoA (salicyloyl-CoA) are probably monooxygenated but not hydrogenated. The purified enzyme is a homodimer and has been resolved preparatively into three major species by anion-exchange chromatography on Mono Q. All three species have the same specific activity when reconstituted to full content of FAD, they differ, however, substantially in their mode of binding FAD. The oxidized or fully reduced enzyme forms bind tightly 0.5 mol/mol of the substrate 2-aminobenzoyl-CoA (Kd = 1-2 microM). The enzyme can be depleted of approximately 50% of its FAD, which corresponds to essentially complete removal from one of the two binding sites, reflecting a large difference in the affinity for FAD. From this it is deduced that the two sites are not equivalent. Removal of FAD from one binding site leads to loss of the hydrogenation capacity of the enzyme, while monooxygenation catalysis is retained. The FAD cofactors of the two binding sites differ drastically in their reactivities towards NADH, oxygen and N-ethylmaleimide. Exchange of reducing equivalents between the two FAD cofactors at the respective binding sites is very slow and irrelevant compared to the rates of catalysis. It is concluded that the enzyme, which has been proposed to consist of two identical polypeptide chains [Altenschmidt, U., Bokranz, M. & Fuchs, G. (1992) Eur. J. Biochem. 207, 715-722], contains two active centers which differ substantially in their catalytic activity. One center belongs to the class of monooxygenases, the other one to the (de)hydrogenases. This must result from substantially different interaction of the same flavin cofactors with protein functional groups and is, to our knowledge, unprecedented in flavoprotein enzymology.

Catalysis↗
PubMed1995

Effects of two mutations detected in medium chain acyl-CoA dehydrogenase (MCAD)-deficient patients on folding, oligomer assembly, and stability of MCAD enzyme.

We have used expression of human medium chain acyl-CoA dehydrogenase (MCAD) in Escherichia coli as a model system for dissecting the molecular effects of two mutations detected in patients with MCAD deficiency. We demonstrate that the R28C mutation predominantly affects polypeptide folding. The amounts of active R28C mutant enzyme produced could be modulated between undetectable to 100% of the wild-type control by manipulating the level of available chaperonins and the growth temperature. For the prevalent K304E mutation, however, the amounts of active mutant enzyme could be modulated only in a range from undetectable to approximately 50% of the wild-type, and the assembled mutant enzyme displayed a decreased thermal stability. Two artificially constructed mutants (K304Q and K304E/D346K) yielded clearly higher amounts of active MCAD enzyme than the K304E mutant but were also responsive to chaperonin co-overexpression and growth at low temperature. The thermal stability profile of the K304E/D346K double mutant was shifted to even lower temperatures than that of the K304E mutant, whereas that of the K304Q mutant was closely similar to the wild-type. Taken together, the results show that the K304E mutation affects (i) polypeptide folding due to elimination of the positively charged lysine and (ii) oligomer assembly and stability due to replacement of lysine 304 with the negatively charged glutamic acid.

Acyl-CoA Dehydrogenase↗
PubMed1994

Characterization of wild-type human medium-chain acyl-CoA dehydrogenase (MCAD) and mutant enzymes present in MCAD-deficient patients by two-dimensional gel electrophoresis: evidence for post-translational modification of the enzyme.

Two-dimensional gel electrophoresis was used to study and compare wild-type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and mis-sense mutant enzyme found in patients with MCAD deficiency. By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wild-type protein. After expression of the cDNA in eukaryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E. coli and another one shifted to lower pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer. Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild-type protein, but is degraded much more readily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed. For K304E mutant MCAD, the nonmodified spot is relatively stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl-CoA Dehydrogenase↗
PubMed1993

Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction.

The bioluminescent reaction of luciferase isolated from Vibrio harveyi, strain M17, was initiated by mixing the luciferase-bound flavin 4a-hydroperoxide intermediate, purified in advance, with a long-chain aldehyde (dodecanal or octanal) at -4 degree C. Measurements of absorbance changes from 300 to 600 nm during the course of the reaction revealed the existence of three sets of isosbestic points and three kinetic phases, the second of which parallels kinetically the decay of bioluminescence, measured concurrently. The absorbance changes in this second step and the decay of light emission exhibited similar deuterium isotope effects; this is postulated to be the step giving rise to the excited state and the enzyme-bound flavin 4a-hydroxide. The first step of the reaction, however, did not show an isotope effect; the intermediate thereby formed, observed here for the first time, is postulated to correspond to the luciferase-bound flavin 4a-peroxyhemiacetal.

Flavins↗
PubMed1993

Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation.

The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation. Depending on which of the three amino acids--lysine (wild-type), glutamic acid (K304E) or glutamine (K304Q) are present at position 304 of the mature polypeptide, three different patterns were observed in our assay system: (i) solubility, tetramer formation and yield of enzyme activity of wild-type MCAD is largely independent of GroESL co-overexpression; (ii) the larger part of the K304Q mutant is insoluble without and solubility is enhanced with GroESL co-overexpression; solubility correlates with the amount of tetramer detected and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected in Western blotting. In a first attempt to estimate the specific activity, we show that tetrameric K304E and K304Q mutant MCAD display a specific activity in the range of the wild-type enzyme. Taken together, our results strongly suggest, that the K304E mutation primarily impairs the rate of folding and subunit assembly. Based on the data presented, we propose that lysine-304 is important for the folding pathway and that an exchange of this amino acid both to glutamine or glutamic acid leads to an increased tendency to misfold/aggregate. Furthermore, exchange of lysine-304 with an amino acid with negative charge at position 304 (glutamic acid) but not with a neutral charge (glutamine) negatively affects conversion to active tetramers. A possible explanation for this latter effect--charge repulsion upon subunit docking--is discussed.

Acyl-CoA Dehydrogenase↗
PubMed1993

Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis.

The reaction of two D-amino acid oxidases from the yeasts Rhodotorula gracilis and Trigonopsis variabilis with the substrates alanine and valine in their 2-1H and 2-2H forms was studied employing the stopped-flow spectrophotometric technique. The turnover numbers at infinite substrate and oxygen concentrations were: 20,700/4,250 and 1,730/360 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,150/440 and 2,500/520 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The rates of anaerobic enzyme flavin reduction were 20,100/4,000 and 1,820/350 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,470/350 and 2,460/480 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The isotope effects on enzyme reduction were 5.0 and 5.2 for Rhodotorula and 9.9 and 5.1 for Trigonopsis D-amino acid oxidases with alanine and valine, respectively. This suggests that the intrinsic isotope effect on rupture of the substrate alpha-C-H bond can be as high as 10. The rate-determining step corresponds to the enzyme reductive half-reaction in contrast to the mammalian kidney enzyme where it is the product release from oxidized enzyme (Massey, V., and Gibson, Q.H. (1964) Fed. Proc. 23, 18-29). Upon anaerobic reaction with substrate, the yeast enzymes do not form the transient long wavelength absorbing species which are characteristic of the mammalian protein. This is due only in part to rapid dissociation of iminoacid product and is ascribed to intrinsic differences between the charge-transfer complexes of reduced enzyme flavin and product of the yeast as compared to the mammalian enzyme. With the Trigonopsis enzyme the flavin radical anion appears to be strongly stabilized and can be produced quantitatively.

Alanine↗
PubMed1993

Phenylalanine hydroxylase-stimulating protein/pterin-4 alpha-carbinolamine dehydratase from rat and human liver. Purification, characterization, and complete amino acid sequence.

Phenylalanine hydroxylase-stimulating protein, also known as pterin-4 alpha-carbinolamine dehydratase (PHS/PCD), was purified from rat and, for the first time, from human liver. We obtained their complete protein primary sequence using a combination of liquid secondary ionization mass spectrometry/tandem quadrupole mass spectrometry, electrospray ionization mass spectrometry, and Edman microsequence analysis. The amino acid sequences of human and rat PHS/PCD were found to be identical. Surprisingly, the primary structure of PHS/PCD is also essentially identical to a protein of the cell nucleus, named dimerization cofactor of hepatocyte nuclear factor 1 alpha, recently reported to be involved in transcription (Mendel, D. M., Khavari, P. A., Conley, P. B., Graves, M. K., Hansen, L. P., Admon, A., and Crabtree, G. R. (1991) Science 254, 1762-1767).

Amino Acid Sequence↗
PubMed1993

Mechanism of bacterial bioluminescence: 4a,5-dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics.

Bioluminescence catalyzed by bacterial luciferases was measured using FMN, iso-FMN (6-methyl-8-nor-FMN), and FMN analogs carrying the following substituents at position 8: -H, -Cl, -F, SMe, SOMe, -SO2Me, or -OMe. The first-order rate constants for the decay of light emission correlate with the one-electron oxidation potentials of the 4a,5-dihydro forms of the FMN analogs. To determine the values of these potentials, isoalloxazine (flavin) derivatives having the 4a,5-propano-4a,5-dihydro structure and -H, -CH3, -Cl, -OCH3, and -NH2 as substituents at position 8 have been synthesized as models for the 4a-peroxy-4a,5-dihydroflavin intermediates occurring during catalysis by the flavin-dependent monooxygenase luciferase. The tetrahydropyrrole ring between positions 4a and 5 of these isoalloxazine derivatives stabilizes the 4a,5-dihydroflavin by impeding formation of the thermodynamically more stable 1,5-dihydro form. One-electron oxidation potentials (Eobs) were measured by cyclic voltammetry and used to determine the empirical coefficients in the Swain equation. On the basis of this, the one-electron oxidation potentials of 4a,5-propano-4a,5-dihydro analogs with other substituents in position 8 were calculated (Ecalc). The bioluminescence reaction rate is fastest with FMN analogs of lowest oxidation potential; i.e., the slope of the correlation is negative. This indicates that in the rate-limiting step the 4a,5-dihydroflavin moiety donates negative charge. The results are compatible with an intramolecular, chemically initiated electron exchange luminescence mechanism for the bacterial luciferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrochemistry↗
PubMed1993

Characterization of D-amino acid oxidase from Trigonopsis variabilis.

D-amino acid oxidase from Trigonopsis variabilis was purified to homogeneity as a well resolved flavoprotein. Specific activity of pure enzyme was 86.6 U/mg at 30 degrees C and pH 8.5. Optimum pH for enzyme activity was 7.5 and optimum temperature was 55 degrees C. The enzyme is a non-glycosylated homodimer; the protein monomer had a M(r) of 38 +/- 2 kDa and contained one molecule of non covalently bound FAD per mole of monomer. A single molecular form with an isoelectric point of 5.1 was detected in isoelectrofocusing. The A272/A455 ratio as calculated from the absorbance spectrum was 8.4. The enzyme bound competitive inhibitors benzoate and anthranilate giving typical flavin spectral perturbations.

Amino Acid Sequence↗
PubMed1992

Studies on the active centre of Rhodotorula gracilis D-amino acid oxidase and comparison with pig kidney enzyme.

D-Amino acid oxidase (EC 1.4.3.3) from Rhodotorula gracilis has been reconstituted with 8-chloro-, 8-mercapto-, 6-hydroxy-, 2-thio-, 5-deaza- and 1-deaza-FAD, and the properties of the resulting complexes have been studied and compared with those of the correspondingly modified pig kidney D-amino acid oxidases. Binding appears to be tight for most analogues, at least as tight as for native FAD (approximately 10(-8) M). 8-Mercapto- and 6-hydroxy-FAD bind in their para- and ortho-quinoid forms respectively to yeast D-amino acid oxidase, inferring the presence of a positive charge near the flavin N(1) position, as in the case of the mammalian enzyme. On the other hand, important differences in active-site microenvironment emerge: solvent accessibility to flavin position 8 is drastically restricted in yeast D-amino acid oxidase as indicated by the unreactivity of 8-chloro- and 8-mercapto-FAD enzyme with thiolates and alkylating agents. Significantly different microenvironments are also likely to occur around the flavin positions N(1)-C(2) = 0, N(3)-H and N(5). This is deduced from the differences in interaction of the two proteins with 1-deaza-FAD, 5-deaza-FAD and 2-thio-FAD and from the properties of the respective complexes. The same re-side flavin stereospecificity as shown by the mammalian enzyme was determined for the yeast enzyme using 8-hydroxy-5-deaza-FAD. Thus we can deduce the presence of a similar pattern of functional groups at the active centres of the two enzymes, while the fine tuning of specificity and regulation correlate with environmental differences at specific flavin loci.

Animals↗
PubMed1992

7-substituted pterins in humans with suspected pterin-4a-carbinolamine dehydratase deficiency. Mechanism of formation via non-enzymatic transformation from 6-substituted pterins.

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7-substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L-tetrahydrobiopterin as 7-(1',2'-dihydroxypropyl)pterin was proven by gas-chromatography mass spectrometry. Tetrahydroneopterin and 6-hydroxymethyltetrahydropterin also are converted to their corresponding 7-substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6-substituted pterin to 7-substituted pterin conversion occurs in the absence of pterin-4a-carbinolamine dehydratase and is shown to be a nonenzymatic process. 7-Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6-tetrahydrobiopterin (Ki approximately 8 microM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7-substituted pterins from their 6-substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine.

Biotransformation↗
PubMed1992

Purification and properties of prostaglandin 9-ketoreductase from pig and human kidney. Identity with human carbonyl reductase.

Prostaglandin 9-ketoreductase (PG-9-KR) was purified from pig kidney to homogeneity, as judged by SDS/PAGE using an improved procedure. The enzyme is pro-S stereoselective with regard to hydrogen transfer from NADPH with prostaglandin E2 as substrate and reduces its 9-keto group with approximately 90% stereoselectivity to form prostaglandin F2 alpha. Approximately 8% of the prostaglandin F formed has the beta-configuration. In addition to catalyzing the interconversion of prostaglandin E2 to F2 alpha, PG-9-KR also oxidizes prostaglandin E2, F2 alpha and D2 to their corresponding, biologically inactive, 15-keto metabolites. Incubation of PG-9-KR with prostaglandin F2 alpha and NAD+ leads to the preferential formation of 15-keto prostaglandin F2 alpha rather than prostaglandin E2. This suggests that the prostaglandin E2/prostaglandin F2 alpha ratio is not determined by the NADP+/NADPH redox couple. The enzyme also reduces various other carbonyl compounds (e.g. 9,10-phenanthrenequinone) with high efficiency. The catalytic properties measured for PG-9-KR suggest that its in vivo function is unlikely to be to catalyze formation of prostaglandin F2 alpha. The monomeric enzyme has a molecular mass of 32 kDa and exists as four isoforms, as judged by isoelectric focusing. PG-9-KR contains 1.9 mol Zn2+/mol enzyme and no other cofactors. Human kidney PG-9-KR was also purified to homogeneity. The human enzyme has a molecular mass of 34 kDa and also exists as four isoforms. Polyclonal antibodies raised against pig kidney PG-9-KR cross-react with human kidney PG-9-KR and also with human brain carbonyl reductase, as demonstrated by Western blot analysis. Sequence data of tryptic peptides from pig kidney PG-9-KR show greater than 90% identity with human placenta carbonyl reductase. From comparison of several properties (catalytical, structural and immunological properties), it is concluded that PG-9-KR and carbonyl reductase are identical enzymes.

Alcohol Oxidoreductases↗